Recombinant alkaline phosphatase mutant and its preparation method

Abstract

This application discloses a recombinant alkaline phosphatase mutant and its preparation method. In this application, alkaline phosphatase derived from Shewanella sp. is used as the target for modification. Site-directed mutagenesis of the amino acid sequence is performed to enhance the thermosensitivity of the alkaline phosphatase, making it suitable for detection by flight mass spectrometry. After the reaction, the enzyme can be inactivated by a short heating period. The alkaline phosphatase mutant provided in this application is easily expressed in a prokaryotic expression system, reducing costs and making it suitable for mass production.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and in particular to a recombinant alkaline phosphatase mutant and a preparation method thereof. Background Art

[0002] Alkaline phosphatase (AP, E 3.1.3.1) is a nonspecific phosphomonoesterase and a homodimeric metalloproteinase. It catalyzes the hydrolysis of phosphate monoesters to form inorganic phosphate and the corresponding alcohols, phenols, or sugars. In the presence of high concentrations of phosphate acceptors, AP catalyzes phosphate group transfer reactions. Alkaline phosphatases are widely found in nature. While AP enzymes from different sources vary significantly in molecular weight and sequence, the active site is highly conserved. Each AP monomer possesses an active center composed of an aspartate-serine-alanine triplet, arginine, water molecules, metal ions, and its ligand amino acids. The Escherichia coli AP enzyme is the most thoroughly studied. Due to its well-defined catalytic mechanism and broad substrate reactivity, it is widely used as a signaling enzyme in medicine, immunology, and analytical biotechnology.

[0003] In the human body, AP enzyme, as a key hydrolase in the phosphate metabolic pathway, catalyzes the hydrolysis and dephosphorylation of various phosphate esters in proteins, nucleic acids, and small molecules. Therefore, in clinical medicine, the detection of AP activity in serum is often used as an important means of diagnosing and detecting diseases. For example, the detection of serum bone AP enzyme can provide early diagnosis of bone metabolic diseases, detect treatment effects, and predict disease prognosis. In immunological research, enzyme-linked immunosorbent assay (ELISA) uses AP enzyme-labeled antibodies for reaction. That is, the detected substance mainly interacts with the substrate through the color development after the reaction of alkaline phosphatase with the color developer or the luminescent substrate after dephosphorylation, which has better stability and sensitivity. At the same time, alkaline phosphatase can catalyze the removal of the 5' terminal phosphate group of DNA molecules, thereby preventing the carrier from self-ligating, and can replace isotope-labeled nucleotide probes for molecular hybridization.

[0004] Among the common alkaline phosphatase products currently on the market, calf intestinal alkaline phosphatase (MAP) has the highest activity, approximately 20-40 times that of Escherichia coli alkaline phosphatase (EAP). However, MAP has poor thermal stability and requires Mg2+ activation. While E. coli expression is low-cost and has good thermal stability, the product activity is relatively low. Therefore, the current mainstream research direction is to obtain highly active EAP enzymes while maintaining good thermal stability. In the existing technology, although some researchers have improved enzyme activity through mutation modification, the modified bacteria are not suitable for large-scale production. After the modified EAP enzyme-engineered bacteria are cultured in an expanded volume, the expression level of the target protein is significantly reduced, which restricts the production and application of AP enzymes. Summary of the Invention

[0005] The object of the present invention is to provide a thermosensitive alkaline phosphatase mutant.

[0006] Another object of the present invention is to provide a nucleic acid molecule.

[0007] Another object of the present invention is to provide a vector compatible with the polynucleotide sequence encoding alkaline phosphatase and its mutants.

[0008] Another object of the present invention is to provide a method for preparing alkaline phosphatase and its mutants.

[0009] Another object of the present invention is to provide a kit containing a polynucleotide sequence encoding alkaline phosphatase and its mutants.

[0010] To solve the above technical problems, the first aspect of the present invention provides an alkaline phosphatase mutant, wherein the alkaline phosphatase mutant undergoes a mutation at one or more sites selected from the following group in the wild-type alkaline phosphatase sequence: W 348, Q349, V 365, S 369 and R 372; wherein the amino acid residue numbering adopts the numbering shown in SEQ ID NO.7.

[0011] In some preferred embodiments, the amino acid sequence of the wild-type alkaline phosphatase is shown in SEQ ID NO.:7.

[0012] In some preferred embodiments, the number of mutation sites is 1 to 5, for example, 1, 2, 3, 4 or 5.

[0013] In some preferred embodiments, the amino acid sequence of the alkaline phosphatase mutant has at least 80% homology with SEQ ID NO.7; more preferably, it has at least 90% homology; most preferably, it has at least 95% homology; such as at least 96%, 97%, 98%, or 99% homology.

[0014] In some preferred embodiments, the alkaline phosphatase mutant is mutated based on the wild-type alkaline phosphatase shown in SEQ ID NO.: 7, and the alkaline phosphatase mutant includes a mutation site selected from the following group: W 348A, Q349A, V 365A, S 369A and R 372A.

[0015] In some preferred embodiments, the alkaline phosphatase mutant is selected from the following group:

[0016]

[0017] In some preferred embodiments, the amino acid sequence of the alkaline phosphatase is selected from the following group:

[0018] (i) the amino acid sequence shown in SEQ ID NO. 1-6; and

[0019] (ii) a polynucleotide having a homology greater than 95% with the amino acid sequence shown in SEQ ID NO. 1-6.

[0020] The second aspect of the present invention provides a polynucleotide molecule, which encodes the alkaline phosphatase mutant described in the first aspect of the present invention.

[0021] The third aspect of the present invention provides a vector, characterized in that the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0022] The fourth aspect of the present invention provides a host cell, characterized in that the host cell contains the vector described in the third aspect of the present invention or the chromosome is integrated with the nucleic acid molecule described in the second aspect of the present invention.

[0023] In some preferred embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0024] In some preferred embodiments, the host cell is Escherichia coli.

[0025] In some preferred embodiments, the host cell is Escherichia coli BL21 (DE3) strain.

[0026] In some preferred embodiments, the eukaryotic cell is a yeast cell.

[0027] The fifth aspect of the present invention provides a method for preparing the alkaline phosphatase mutant according to the first aspect of the present invention, characterized in that it comprises the steps of:

[0028] (i) culturing the host cell according to the fourth aspect of the present invention under suitable conditions to express the alkaline phosphatase mutant; and

[0029] (ii) isolating the alkaline phosphatase mutant.

[0030] In some preferred embodiments, the host cells are cultured using SB, TB, LB, or SOC culture media, more preferably using TB and LB culture media, and most preferably using TB culture media.

[0031] In some preferred embodiments, the host cell is cultured at a temperature of 16 to 19°C or 35 to 39°C.

[0032] The sixth aspect of the present invention provides a kit, characterized in that the kit comprises the alkaline phosphatase mutant described in the first aspect of the present invention.

[0033] Compared with the prior art, the present invention has at least the following advantages:

[0034] (1) The present invention uses alkaline phosphatase derived from Shewanella sp. as the modification target, performs site-directed mutagenesis on the amino acid sequence, improves the thermosensitivity of alkaline phosphatase, and makes it suitable for detection by flight mass spectrometry. After the reaction, the enzyme can be inactivated by a short heating time.

[0035] (2) The alkaline phosphatase mutant provided by the present invention is easy to express in a prokaryotic expression system, which reduces costs and is suitable for mass production.

[0036] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail 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 listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] One or more embodiments are exemplarily described by the figures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments.

[0038] Figure 1 3D structure diagram of alkaline phosphatase derived from Shewanella sp. according to an embodiment of the present invention;

[0039] Figure 2 Schematic diagram of the structural alignment of alkaline phosphatase from Shewanella sp and E. coli according to an embodiment of the present invention;

[0040] Figure 3 This is a partial enlarged view of the structure alignment of alkaline phosphatase derived from Shewanella sp according to an embodiment of the present invention;

[0041] Figure 4 This is a diagram showing the identification results of a small-scale expression of alkaline phosphatase in Escherichia coli according to an embodiment of the present invention;

[0042] Figure 5 This is an electrophoretic diagram of nickel column purification of alkaline phosphatase according to an embodiment of the present invention;

[0043] Figure 6 This is an electrophoretic diagram of alkaline phosphatase purified by nickel column QP column according to an embodiment of the present invention;

[0044] Figure 7 is a standard curve diagram for determining alkaline phosphatase / mutant enzyme activity according to an embodiment of the present invention;

[0045] Figure 8 is a graph showing the thermal sensitivity of alkaline phosphatase / mutants according to an embodiment of the present invention. DETAILED DESCRIPTION

[0046] In the prior art, alkaline phosphatases with good heat resistance (inactivated by treatment at 65°C for 5 minutes) are mainly derived from bovine small intestine and shrimp. Bovine small intestine alkaline phosphatases are mostly extracted and purified from natural products, while shrimp alkaline phosphatases are mostly expressed in yeast. Prokaryotic expression of alkaline phosphatases from Escherichia coli (E. coli) and marine efficient phosphate removal bacteria (Shewanella sp) has high activity, but poor heat resistance and unsatisfactory heat inactivation at 65°C. Through extensive and in-depth research, the inventors unexpectedly discovered a mutant of alkaline phosphatase, as described in the present invention, that exhibits good heat sensitivity while retaining good enzymatic activity.

[0047] Alkaline phosphatase mutants

[0048] In the present invention, the target protein is alkaline phosphatase from Shewanella sp (amino acid sequence shown in SEQ ID NO. 7). The mutant is obtained by mutating the alkaline phosphatase via a predetermined mutation pattern, which includes mutations at one or more sites selected from the group consisting of W 348, Q 349, V 365, S 369, and R 372. Preferably, the amino acids selected from the mutation sites are mutated to alanine. The predetermined mutation patterns include any one or a combination of W 348A, Q 349A, V 365A, S 369A, and R 372A. When at least two, more preferably at least three, more preferably at least four, and even more preferably all of the amino acids at these mutation sites are mutated to alanine (A), the target protein is obtained.

[0049] In some embodiments of the present invention, the amino acid sequence of the alkaline phosphatase mutant is selected from any one of the following,

[0050] (i) the amino acid sequence shown in SEQ ID NO. 1-6; and

[0051] (ii) a polynucleotide having a homology greater than 95% with the amino acid sequence shown in SEQ ID NO. 1-6.

[0052] The target protein of the present invention can be prepared by conventional methods in the art, such as site-directed mutagenesis. One or more bases in a target nucleic acid fragment or plasmid are replaced with other bases using methods such as polymerase chain reaction (PCR). The plasmid containing the mutated nucleic acid fragment is transformed into Escherichia coli to obtain transformants, which are then cultured to obtain mutants. Mutants are obtained by site-directed mutagenesis using a commercially available site-directed mutagenesis kit.

[0053] Nucleic acid sequence encoding target protein mutant

[0054] In the present invention, the full-length nucleotide sequence of the target protein mutant or its element or its fragment can usually be obtained by PCR amplification, recombinant method or artificial synthesis method. For PCR amplification, primers can be designed based on the disclosed relevant nucleotide sequence, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify and obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications and then splice the fragments amplified each time together in the correct order. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant method. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the host cells after the proliferation by conventional methods.

[0055] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0056] Methods using PCR technology to amplify DNA / RNA are preferably used to obtain the genes 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. Amplified DNA / RNA fragments can be separated and purified using conventional methods, such as by gel electrophoresis.

[0057] Synonymous codon preference optimization

[0058] To overcome the potential problem of reduced production when expressing heterologous proteins in host cells, the present invention relates to polynucleotide sequences optimized for synonymous codon preference. The obtained target gene sequence is subjected to synonymous codon preference optimization, and the gene's synonymous codons are adjusted according to the host's codon preference, thereby eliminating rare codons and improving the expression efficiency of the heterologous gene. The target gene sequence optimized for synonymous codon preference can express the same amino acid sequence as the target protein. In an embodiment of the present invention, the present invention relates to optimized codon sequences obtained by codon optimization of gene sequences encoding alkaline phosphatase mutants, as shown in SEQ ID NOs:8-13. The present invention also relates to polynucleotides having a homology greater than 80%, preferably greater than 85%, more preferably greater than 90%, more preferably greater than 91%, and more preferably greater than 95% to the sequences shown in SEQ ID NOs:8-13; and polynucleotides complementary to the sequences shown in SEQ ID NOs:8-13.

[0059] Target gene vector

[0060] The present invention also relates to vectors comprising the polynucleotides of the present invention. As used herein, "vector" refers to a linear or circular DNA molecule comprising a segment encoding a protein of interest operably linked to other segments that provide for its transcription. Such additional segments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, vectors, and the like. The vector segment may be derived from a host organism, another organism, a plasmid, or viral DNA, or may be synthetic. The vector may be synthetic or any expression vector that is readily amenable to recombinant DNA manipulations, the choice of vector generally depending 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 that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the host cell genome and replicates along with the chromosome into which it is integrated. In one embodiment, the vector of the present invention is an expression vector. In one embodiment of the present invention, pET-28a(+) was selected as the vector to achieve more efficient expression efficiency.

[0061] 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 appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Illustratively, a DNA endonuclease is used to cut the vector DNA molecule into a linear molecule that can be connected to the exogenous gene, and then the codon-optimized target gene fragment is connected to the vector. The insertion of the exogenous DNA fragment can be achieved by selectively ligating the exogenous DNA fragment with a single restriction endonuclease, directional cloning of double restriction endonuclease fragments, ligating the exogenous DNA fragment with different restriction endonuclease sites, blunt end ligation, artificial linker ligation, or oligonucleotide end ligation.

[0062] The vector containing the target gene is transformed into the host cell

[0063] The present invention also relates to host cells produced by genetic engineering using the vectors or fusion protein coding sequences of the present invention. A vector containing a codon-optimized target gene can be inserted, transfected, or otherwise transformed into a host cell by known methods, thereby obtaining a transformant containing the codon-optimized target gene of the present invention and capable of expressing the target protein. In the present invention, a "host cell" is a cell into which an exogenous polynucleotide and / or vector has been introduced. The host cell can be a eukaryotic host cell or a prokaryotic host cell. The host cell is preferably a bacterium, and is preferably Escherichia coli, more preferably Escherichia coli Rosetta (DE3) strain.

[0064] Method for preparing target protein

[0065] The present invention also relates to a method for preparing a target protein, which can be expressed or produced using the polynucleotide sequence of the present invention. Generally, the method comprises the following steps:

[0066] (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0067] (2) host cells cultured in a suitable culture medium;

[0068] (3) Isolate and purify proteins from culture medium or cells.

[0069] Among them, in step (1), the transformation or transduction of a suitable host cell with the recombinant expression vector containing the polynucleotide can be carried out by conventional techniques well known to those skilled in the art. When the host is Escherichia coli, heat shock method and electroporation method can be used.

[0070] The transformant obtained can be cultivated with a conventional method, and expresses the polypeptide encoded by the gene of the present invention. According to the host cell used, the culture medium used in the cultivation can be selected from various conventional culture media, and is preferably SB, TB, LB or SOC culture medium. Under the condition that is suitable for host cell growth, cultivate. After the host cell grows to a suitable cell density, induce the promoter selected with a suitable method (such as temperature conversion or chemical induction), and the cell is cultivated for a period of time. For promoting the expression of the target protein and promoting the expression of soluble protein, a preferred embodiment of the present invention uses the host cell cultivated with TB or LB culture medium, and contains the kanamycin resistance gene in the culture medium used.

[0071] In order to further promote the soluble expression of the target protein, in a preferred embodiment of the present invention, the host cells are cultured to an OD 600After the pH value reaches 0.6-0.8, IPTG is used for induction and the culture is continued at 17 to 19°C or 35 to 39°C for about 8 to 12 hours.

[0072] The protein in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the protein can be separated and purified by various separation methods using its physical, chemical and other properties. Therefore, in the present invention, after successfully culturing to obtain the target protein, it is also related to the steps of separating and purifying it, such as in step (3), separating and purifying the protein from the culture medium to obtain a high-purity target protein. Although the method for purifying the target protein is conventional means well known to those skilled in the art, it includes but is not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0073] The purified target protein product is dialyzed and the dialyzed sample is collected. The concentration of the dialyzed sample can be measured by BCA assay to calculate the yield.

[0074] In the present invention, any exemplary or exemplary wording (e.g., "") provided for certain embodiments herein is used only to better present the present invention and does not limit the scope of the present invention claimed in other ways. Any wording herein should not be interpreted as indicating an element not described in the claims that is indispensable for the implementation of the present invention.

[0075] If a definition or use of a term in a referenced document is inconsistent or inconsistent with the definition of that term as described herein, the definition of that term as described herein applies and the definition of that term in the referenced document does not apply.

[0076] Various terms are used herein as follows. If a term used in a claim is not defined below, it should be given the broadest definition persons in the art have given that term as reflected in printed publications or issued patents at the time of filing.

[0077] As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., nucleic acid or polypeptide) present in its natural source. In one embodiment, the nucleic acid or polypeptide is found only in the presence of solvents, buffers, ions, or other components that are normally present in a solution thereof, if any. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.

[0078] As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0079] The present invention also relates to variants of the aforementioned polynucleotides, which encode protein fragments, analogs, and derivatives having the same amino acid sequence as the present invention. These polynucleotide variants may 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 an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded polypeptide.

[0080] As used herein, the term "codon optimization" refers to a method for improving gene synthesis efficiency by avoiding the use of low-utilization or rare codons based on the differences in codon usage exhibited by organisms actually expressing or producing proteins (including Escherichia coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

[0081] As used herein, the terms "homology" and "identity" are used interchangeably and refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be measured by the following method. The nucleotide or amino acid sequence of a polynucleotide or polypeptide is arranged, and the number of positions containing identical nucleotides or amino acid residues in the arranged polynucleotide or polypeptide is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the arranged polynucleotide or polypeptide. A polynucleotide can be different in one position, for example, according to comprising different nucleotides (i.e., replacement or variation) or deletions of nucleotides (i.e., insertion or deletion of one or two nucleotides in a polynucleotide). A polypeptide can be different in one position, for example, by containing an amino acid (i.e., replacement or variation) or deletion of an amino acid (i.e., insertion of an amino acid or amino acid deletion in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of amino acid residues in a polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0082] 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 of the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complement sequence is GTTCAT.

[0083] 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. After expression, the host cells or expression products may be harvested, i.e., recovered.

[0084] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0085] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0086] Example 1

[0087] In this example, the modified regions and modification strategies for alkaline phosphatase were determined by three-dimensional structural comparison, and several modified alkaline phosphatase mutants were obtained. The specific steps are as follows:

[0088] (1) Developing transformation strategies by comparing three-dimensional structures

[0089] The alkaline phosphatase from Shewanella sp was selected as the transformation object, and the three-dimensional structure modeling was carried out. Alphafold2 was used to establish the three-dimensional structure of Shewanella sp (such as Figure 1 ). The structures of alkaline phosphatases from Shewanella sp and E. coli were compared, and the comparison results are as follows: Figure 2 shown.

[0090] according to Figure 2 The following conclusions can be drawn:

[0091] First, R166 and D153 are active site amino acids. Figure 2Orange represents SAP from Shewanella sp., light cyan represents SAP from E. coli, and green represents the substrate, including Zn, Mg, and PO₄ molecules. Structural comparison revealed that the active sites of alkaline phosphatases from Shewanella sp. and E. coli are highly conserved. The two amino acids in red are R166 and D153, which bind to the substrate and are preliminarily identified as important active site amino acids.

[0092] Second, the four helices at amino acids 330-390 are the key areas for modification. Figure 3 As shown, the differences between Shewanella sp.-derived SAP and E. coli-derived SAP are mainly in the four helices at amino acids 330-390 ( Figure 3 Multiple amino acids in the two helices participate in intramolecular interactions. These sites are distant from the active center, and mutants may weaken or disrupt intramolecular helical formation, thereby reducing thermal stability. Mutations at amino acid residues 330-390 were constructed, and the vast majority of mutants showed very low expression activity, requiring further testing to identify the mutation sites.

[0093] (2) Testing to determine the mutation site

[0094] Some of the mutants obtained after testing are shown in Table 1 below:

[0095] Table 1

[0096] Amino acid site Amino acid name Mutable amino acids 348 Trp(W) Ala(A) 349 Gln(Q) Ala(A) 365 Val(V) Ala(A) 369 Ser(S) Ala(A) 372 Arg(R) Ala(A)

[0097] The test results show that among the mutation sites listed in Table 1, Trp348 and Arg372 are extremely important and can therefore be used as priority modification sites.

[0098] (3) Determine the sequence of the alkaline phosphatase mutant

[0099] As shown in Table 2, sequences 1-5 are single-point mutants at Trp 348, Gln 349, Val 365, Ser 369, and Arg 372, respectively, and sequence 6 is a multiple-point mutant (Trp348 / Gln 349 / Val365 / Ser 369 / Arg372).

[0100] Table 2

[0101] Serial number name SEQ ID NO.1 Alkaline phosphatase mutant A-1 SEQ ID NO.2 Alkaline phosphatase mutant A-2 SEQ ID NO.3 Alkaline phosphatase mutant A-3 SEQ ID NO.4 Alkaline phosphatase mutant A-4 SEQ ID NO.5 Alkaline phosphatase mutant A-5 SEQ ID NO.6 Alkaline phosphatase mutant B-1 SEQ ID NO.7 Alkaline phosphatase before mutation

[0102] SEQ ID NO.1:

[0103] MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSPELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTMGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQAQADLARGLGFELNADEVTQLSTARMQGLETMTEAIRKIIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

[0104] SEQ ID NO.2:MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSPELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTMGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWAADLARGLGFELNADEVTQLSTARMQGLETMTEAIRKIIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

[0105] SEQ ID NO.3:

[0106] MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSPELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTMGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWQADLARGLGFELNADEATQLSTARMQGLETMTEAIRKIIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

[0107] SEQ ID NO.4:

[0108] MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSPELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTMGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWQADLARGLGFELNADEVTQLATARMQGLETMTEAIRKIIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

[0109] SEQ ID NO.5:

[0110] MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSPELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTMGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWQADLARGLGFELNADEVTQLSTAAMQGLETMTEAIRKIIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

[0111] SEQ ID NO.6:

[0112] MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSPELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTMGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQAAADLARGLGFELNADEATQLATAAMQGLETMTEAIRKIIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

[0113] SEQ ID NO.7:

[0114] MSVTKTSLLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSY NGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSPELLEKFTAKGYQHISRFEDLATITQPKVIGLFA QVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTMGEMDEFANALEVVEQFVRQHPDTLMMVATADHNTGGLSIGAGGDYRWNPEILRNMS ASTDTLALAALGGDQWQADLARGLGFELNADEVTQLSTARMQGLETMTEAIRKIIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPPKKAKTE

[0115] Example 2

[0116] In this example, based on the alkaline phosphatase mutant sequence obtained in Example 1 and the unmutated Shewanella sp alkaline phosphatase sequence, an adapted prokaryotic expression method was developed to obtain several alkaline phosphatase mutants and alkaline phosphatase expressed in Escherichia coli. The specific steps are as follows:

[0117] (1) Construction of alkaline phosphatase mutant plasmid

[0118] According to the amino acid sequence of Shewanella sp alkaline phosphatase, the N-terminal signal peptide aa1-23 was removed to obtain the sequence shown in SEQ ID NO.7. The corresponding gene sequence was obtained by analyzing SEQ ID NO.7, and the Escherichia coli synonymous codon preference was optimized to obtain optimized codon I. The optimized codon I was ligated into the vector pET-28a(+), and the recombinant plasmid C was synthesized by entrusting Suzhou Jinweizhi Biotechnology Co., Ltd.

[0119] Based on the sequences of several Shewanella sp alkaline phosphatase mutants obtained in Example 1, the corresponding gene sequences were analyzed and optimized for the synonymous codon preference of Escherichia coli, respectively, to obtain the optimized codons shown in Table 3. These optimized codons were ligated into the vector pET-28a(+), and Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize several recombinant plasmids.

[0120] Table 3

[0121]

[0122]

[0123] Optimized codon I (SEQ ID NO.8):

[0124]

[0125] Optimized codon II-1 (SEQ ID NO.9):

[0126]

[0127] Optimized codon II-2 (SEQ ID NO.10):

[0128]

[0129] Optimized codon II-3 (SEQ ID NO.11):

[0130]

[0131] Optimized codon II-4 (SEQ ID NO.12):

[0132]

[0133] Optimized codon II-5 (SEQ ID NO.13):

[0134]

[0135] Optimized codon III-1 (SEQ ID NO.14):

[0136]

[0137] (2) Introduction of recombinant plasmid into host Escherichia coli

[0138] Take 1 μL of the expression plasmid obtained in step (1) and add it to 30 μL of competent E. coli BL21 (DE3) under ice bath conditions. Place it on ice for 30 minutes, then in a 42°C water bath for 45 seconds. Immediately place it on ice for 2 minutes, add 400 μL of SOC medium without antibiotics, and culture it at 37°C with shaking at 230 rpm for 45 minutes. Take 100 μL of the bacterial solution and evenly spread it on an LB plate containing 100 μg / mL kanamycin resistance and culture it in a 37°C incubator overnight.

[0139] (3) Target gene expression

[0140] The monoclonal clones prepared in step (2) were picked and inoculated into TB and LB culture media containing 100 μg / mL kanamycin resistance aseptically. The culture was shaken at 37°C and 220 rpm until the OD600 was between 0.6 and 0.8. IPTG was used for induction and the culture was shaken at 37°C and 18°C overnight. The samples were ultrasonically broken and identified by SDS-PAGE. For example, the identification results of some samples are shown in Figure 4 . Figure 4 The following figure shows the identification results of the unmutated alkaline phosphatase. It was identified that the product was expressed in the supernatant of TB and LB culture media at 18°C (target band 46 kDa). The product identification results are summarized in Table 4.

[0141] Table 4

[0142]

[0143]

[0144] (4) Purification of expression product (nickel column + QP column)

[0145] Weigh approximately 10g of recombinant cells and resuspend in 50ml of Lysis buffer containing 600mM guanidine hydrochloride using a vortexer. Disrupt cells by sonication: φ10 probe, 10% power, 5.5s on, 9.9s off, for 30min. Centrifuge at 20,000rpm, 4°C for 30min, collect the supernatant, and filter through a 0.22μm membrane. Apply the filtered supernatant to a 5ml Ni column and wash and elute the target protein according to the following steps: Elution Step 1: 0% B, 5CV, Step 2: 0-60% B, 15CV, Step 3: 100% B, 6CV. Flow rate: Load: 2.5ml / min (sample pump), Elution: 5ml / ml, Collection: 8ml / tube (15ml). The collected samples were directly diluted 6-fold with 50mM Tris-HCl, 5% glycerol, pH 7.0 solution and used for Q-HP column purification. A 5ml QP column was used for purification with a mobile phase flow rate of 3ml / min. After loading, 20ml of 50mM Tris-HCl buffer was used to flush the UV and conductivity to baseline. The elution program included: Step 1: 0% B, 8CV, 3ml / min; Step 2: 0-60% B, 20CV, 3ml / min; Step 3: 100% B, 15CV, 3ml / min. The eluted samples were collected for electrophoretic separation. For example, the electropherograms of some samples are shown in Figure 2. Figure 5 and Figure 6 . Figure 5 Lane S: 0.22 μm, Lane F1-3: pass through, Lane 2A1-2B3 gradient elution, Lane 2C3: 100% elution, 10% SDS-PAGE, run: 2.0 μl. Figure 6 In the middle, Lane S:Ni eluted diluted samples, Lane 1A1-2A1 passed through, Lane 2A2-2B3 gradient elution, 2B3: 2.36U / μl, 10% SDS-PAGE, run: 2.0μl.

[0146] The target protein was mainly eluted at 250mM NaCl. F was dialyzed overnight in dialysate and the volume of the collected dialyzed samples was 20ml. The concentration was measured by BCA and the result was: R 2 =0.995, its concentration was 18.2 mg / ml, the yield was 433.2 mg, and the yield was 43.32 mg / g bacteria. The yield and productivity of the purified product are shown in Table 5.

[0147] Table 5

[0148] Codon number Corresponding alkaline phosphatase / mutant Yield mg / g Optimized codon I Unmutated alkaline phosphatase 43.32mg / g Optimized codon II-1 Alkaline phosphatase mutant A-1 35.27mg / g Optimized codon II-2 Alkaline phosphatase mutant A-2 32.46mg / g Optimized codon II-3 Alkaline phosphatase mutant A-3 34.07mg / g Optimized codon II-4 Alkaline phosphatase mutant A-4 28.53mg / g Optimized codon II-5 Alkaline phosphatase mutant A-5 26.37mg / g Optimized codon III-1 Alkaline phosphatase mutant B-1 31.47mg / g

[0149] Example 3

[0150] In this example, the activity of the alkaline phosphatase mutant prepared in Example 2 was determined. The specific steps are as follows:

[0151] (1) Solution preparation

[0152] Prepare 2 mL of working solution according to Table 6 below.

[0153] Table 6

[0154] Reagents Add volume 500mM PNPP 200 μL 10X rCutSmart buffer 200 μL ddH2O 1.6mL

[0155] (2) Activity test

[0156] Preparation of positive alkaline phosphatase (commercially available active alkaline phosphatase): dilute 1 U / μL positive alkaline phosphatase with PBS pH 7.4 buffer (containing 50% glycerol), and then dilute it stepwise according to a gradient, using PBS pH 7.4 buffer as the diluent.

[0157] Instrumental Assay: Preheat the microplate reader to 37°C for 30 minutes. Program the microplate reader: Add 50 μL of the working solution and measure absorbance at 405 nm (A1). Then, add 1 μL of each concentration of positive enzyme solution, react for 5 minutes, and measure absorbance at 405 nm (A2). Calculate the OD difference (A2-A1) between the alkaline phosphatase mutant prepared in Example 2 and the blank, and calculate the enzyme activity, which is summarized in Table 7 below.

[0158] Table 7

[0159]

[0160]

[0161] Example 4

[0162] In this example, the heat sensitivity of the alkaline phosphatase mutant prepared in Example 2 was determined. The specific steps are as follows:

[0163] The alkaline phosphatase mutant prepared in Example 2 was divided into five groups. The four experimental groups were treated at 65, 75, 85, and 95°C for 15 min, respectively; a control group was not heated. The OD values of the samples in each group were then tested. The higher the OD value, the higher the enzyme activity. The OD value of the control group increased with prolonged reaction time, while the OD value of the experimental group remained almost unchanged with prolonged reaction time, indicating that treatment at 65, 75, 85, and 95°C for 15 min could inactivate the mutant enzyme.

[0164] from Figure 8 It can be seen that the alkaline phosphatase mutant can be completely inactivated by short-term high-temperature treatment, and the modified alkaline phosphatase has good heat sensitivity.

[0165] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. An alkaline phosphatase mutant, characterized in that The alkaline phosphatase mutant is mutated based on the wild-type alkaline phosphatase shown in SEQ ID NO.7, and the mutation sites of the alkaline phosphatase mutant are: W 348 A, Q349 A, V 365 A, S 369 A and R 372 A.

2. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the alkaline phosphatase mutant according to claim 1.

3. A carrier, characterized in that The vector contains the polynucleotide molecule according to claim 2.

4. A host cell, characterized in that The host cell contains the vector according to claim 3 or the chromosome has the polynucleotide molecule according to claim 2 integrated therein.

5. A method for preparing the alkaline phosphatase mutant according to claim 1, characterized in that: Including steps: (i) culturing the host cell of claim 4 under suitable conditions to express the alkaline phosphatase mutant; and (ii) isolating the alkaline phosphatase mutant.

6. A kit, characterized in that The kit comprises the alkaline phosphatase mutant according to claim 1.

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