Mutant bst DNA polymerase large fragment, and preparation method therefor

Abstract

Provided are a preparation method and use for a truncated body of a Bst DNA polymerase mutant. The provided truncated body of the Bst DNA polymerase mutant has good heat resistance and can be stably expressed in an E. coli system. The provided preparation method for the truncated body of the Bst DNA polymerase mutant can express a large amount of soluble Bst DNA polymerase.

Description

[Corrected 23.08.2024 according to Rule 26] A mutant Bst DNA polymerase large fragment and its preparation method

[0001] Priority Declaration

[0002] This application claims priority to Chinese patent application No. 2023114455020, filed on November 2, 2023. This application incorporates the entirety of the aforementioned patent application. Technical Field

[0003] The present application relates to the field of biomedicine, and in particular to a truncated form of a Bst DNA polymerase mutant and a preparation method and application thereof. Background Art

[0004] Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus. The gene is 2631 base pairs long, the amino acid sequence is 876 amino acids long, the protein has a molecular weight of 99 kDa, and an isoelectric point of 5.32. The complete Bst DNA polymerase exhibits four activities: 5'-3' exonuclease, 5'-3' DNA polymerase, 3'-5' exonuclease (proofreading), and strand displacement. The C-terminal peptide chain, consisting of amino acids 291-876, performs the remaining three enzymatic activities, excluding the 5'-3' exonuclease activity, and is referred to as the Bst DNA polymerase large fragment. Bst DNA polymerase has an optimal reaction temperature of 65°C and is inactivated above 80°C. Therefore, it cannot be used for thermal cycle sequencing or PCR. It exhibits strong stress resistance, excellent thermal stability, and high tolerance to nonionic surfactants and high salt levels.

[0005] Based on these properties, Bst DNA polymerase has applications in GC-rich DNA sequencing, rapid sequencing of low-content template DNA, isothermal DNA amplification, multiple strand displacement amplification, and whole-genome amplification. Furthermore, Bst DNA polymerase can initiate template-dependent DNA synthesis and randomly add nucleotides to the 3' terminus. In recent years, nucleic acid isothermal amplification technologies have rapidly developed, primarily including rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP). Bst DNA polymerase is a foundational enzyme for both techniques. Because Bst DNA polymerase can perform amplification reactions under constant temperature conditions, ordinary water baths or equipment with a stable heat source can meet the reaction requirements. Compared to conventional PCR testing, it does not rely on expensive PCR instruments and specialized laboratories, and its detection time is shortened (only 1 / 3-1 / 5 of that of PCR technology). This significant advantage has led to its widespread application in various fields, including medical testing, pathogen inspection and quarantine, and food quarantine. Another application direction of Bst DNA polymerase is second-generation sequencing. Also due to its advantage of being able to amplify under constant temperature conditions, it reduces the steps of cycling temperature during library amplification, improves amplification efficiency, and reduces sequencing time. Existing sequencing platforms using Bst DNA polymerase include Roche, Illumina, Solexa, etc.

[0006] Although BstDNA polymerase is commercialized at present, compared with foreign commodity enzyme, domestic market BstDNA polymerase quality is uneven, and localization process is slow, mainly purchased from various foreign biological companies, although the method is simple to operate, quick, but expensive. If BstDNA polymerase gene expression engineering bacteria can be constructed, voluntarily synthesizing BstDNA polymerase, scientific research and production costs can be greatly reduced, and it is also conducive to the localization of BstDNA polymerase. In the prior art, the method for preparing BstDNA polymerase cannot be expressed in supernatant, and purification cost is high, and product activity is poor. Therefore, this area is in urgent need of developing a low-cost method for preparing BstDNA polymerase that is conducive to the high activity of supernatant expression product.

[0007] Summary of the Invention

[0008] The purpose of this application is to provide a truncated form of a Bst DNA polymerase mutant.

[0009] Another object of the present application is to provide a method for preparing a truncated form of a Bst DNA polymerase mutant.

[0010] Another object of the present application is to provide a polynucleotide sequence encoding a truncated form of the Bst DNA polymerase mutant.

[0011] Another object of the present application is to provide a vector compatible with the polynucleotide sequence encoding the truncated form of the Bst DNA polymerase mutant.

[0012] Another object of the present application is to provide a kit containing a polynucleotide sequence encoding a truncated form of a Bst DNA polymerase mutant.

[0013] To solve the above technical problems, the first aspect of the present application provides a truncated form of a Bst DNA polymerase mutant, wherein the amino acid sequence of the truncated form of the Bst DNA polymerase mutant is selected from any one of the following:

[0014] (i) the amino acid sequence of SEQ ID NO. 2; and

[0015] (ii) an amino acid sequence having a homology greater than 95% with the sequence shown in SEQ ID NO. 2.

[0016] In a second aspect of the present application, a polynucleotide encoding a truncated form of a Bst DNA polymerase mutant is provided, wherein the polynucleotide is codon-optimized and selected from any one of the following:

[0017] (i) a polynucleotide having a sequence as shown in SEQ ID NO.1;

[0018] (ii) a polynucleotide having a homology greater than 95% to the sequence shown in SEQ ID NO. 1; and

[0019] (iii) a polynucleotide complementary to the polynucleotide sequence described in (i) or (ii).

[0020] In a third aspect, the present application provides an expression vector comprising the polynucleotide provided in the second aspect of the present application.

[0021] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

[0022] In a fourth aspect, the present application provides a host cell, wherein the host cell comprises the expression vector provided in the third aspect of the present application; or

[0023] The polynucleotide provided in the second aspect of this application is integrated into the genome of the host 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] The fifth aspect of the present application provides a method for preparing a truncated form of a Bst DNA polymerase mutant, the method comprising the steps of: culturing the host cell described in the fourth aspect of the present application to express a target protein; and

[0027] Isolating the target protein to obtain a truncated form of the Bst DNA polymerase mutant;

[0028] In some preferred embodiments, the host cell is obtained by transforming Escherichia coli with a plasmid containing the polynucleotide described in the second aspect of the present application.

[0029] In some preferred embodiments, the host cells are cultured using SB, TB, LB, or SOC culture medium, and more preferably, the host cells are cultured using TB or LB culture medium.

[0030] In some preferred embodiments, the host cells are cultured in a shaking environment.

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

[0032] In some preferred embodiments, when culturing the host cell, the culture medium used contains a kanamycin resistance gene.

[0033] In some preferred embodiments, when culturing the host cells, IPTG is used for induction to express the target protein.

[0034] In some preferred embodiments, when culturing the host cells, the cells are cultured until the OD600 is between 0.6 and 0.8, and then induced with IPTG to express the target protein.

[0035] In some preferred embodiments, the step of separating the target protein comprises:

[0036] The supernatant of the crushed target protein is eluted through the chromatography column simultaneously with the mobile phase, and the eluate is collected.

[0037] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column (Ni-NTA).

[0038] In some preferred embodiments, the mobile phase includes Buffer A, Buffer B and Buffer C, and each Buffer contains sodium phosphate, NaCl and imidazole.

[0039] In some preferred embodiments, the Buffer A contains 20 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole.

[0040] In some preferred embodiments, the Buffer B contains 20 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole.

[0041] In some preferred embodiments, the Buffer A contains 20 mM sodium phosphate and 1 M NaCl.

[0042] In some preferred embodiments, the pH values ​​of Buffer A, Buffer B and Buffer C are all 7.4.

[0043] In some preferred embodiments, the step of isolating the target protein further comprises:

[0044] The collected eluate is treated using an ion exchange column to obtain a treated solution.

[0045] In some preferred embodiments, the step of separating the target protein further comprises: dialyzing the treatment solution.

[0046] In a fifth aspect, the present application provides a kit, comprising: a truncated form of the Bst DNA polymerase mutant provided in the first aspect of the present application; or

[0047] A polynucleotide as provided in the second aspect of this application; or

[0048] As the expression vector provided in the third aspect of this application; or

[0049] The host cell as described in the fourth aspect of the present application.

[0050] Compared with the prior art, this application has at least the following advantages:

[0051] (1) The present application provides a truncated form of a Bst DNA polymerase mutant, which has good heat resistance and can be stably expressed in an Escherichia coli system;

[0052] (2) The method for preparing the truncated form of the Bst DNA polymerase mutant provided in this application can express a large amount of soluble Bst DNA polymerase, has low purification cost, good product activity, and is suitable for industrial production.

[0053] It should be understood that within the scope of this application, the above-mentioned technical features of this application 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

[0054] 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.

[0055] FIG1 is an SDS-PAGE identification diagram of the truncated product of the Bst DNA polymerase mutant according to the embodiment of the present application;

[0056] FIG2 is a nickel column purification result diagram of a truncated Bst DNA polymerase mutant according to an embodiment of the present application;

[0057] FIG3 is a diagram of ion exchange purification of a truncated form of a Bst DNA polymerase mutant according to an embodiment of the present application;

[0058] FIG4 is a standard curve diagram of the enzyme activity test of the truncated form of the Bst DNA polymerase mutant according to the examples of the present application. DETAILED DESCRIPTION

[0059] The Bst DNA polymerases prepared in the prior art have poor activity, low yield, and poor activity. The present applicants, through extensive and in-depth research, obtained mutants and truncated forms of Bst DNA polymerase by mutating and truncating the enzyme. The applicants then analyzed the mutant and / or truncated Bst DNA polymerases to obtain the target gene sequence encoding them. The target gene sequence was optimized to adapt it to the vector and host cell expression system of the present application, resulting in the successful expression of a Bst DNA polymerase with excellent enzymatic activity.

[0060] Bst DNA polymerase mutant

[0061] The present application relates to a Bst DNA polymerase mutant, which is obtained by mutating at least one site selected from the following: K369, A464, and S619 based on the original Bst DNA polymerase sequence. Furthermore, the mutant is obtained by mutating at three sites, K369, A464, and S619, based on the original Bst DNA polymerase sequence. The mutation can be an amino acid substitution, addition, or deletion.

[0062] In some preferred embodiments of the present application, the Bst DNA polymerase mutant is obtained by at least one mutation selected from the following based on the original sequence of the Bst DNA polymerase: K369G, A464G and S619G. Further, the Bst DNA polymerase mutant undergoes K369G, A464G and S619G mutations based on the original sequence of the Bst DNA polymerase, mutating the K at position 369 to G, mutating the A at position 464 to G, and mutating the S at position 619 to G, thereby obtaining the Bst DNA polymerase mutant. The aforementioned mutant can be obtained by conventional in vitro site-directed mutagenesis methods in this area, for example, by introducing desired changes into the target DNA fragment (which can be a genome or a plasmid) through methods such as polymerase chain reaction (PCR), including addition, deletion, and point mutation of bases.

[0063] Truncated forms of Bst DNA polymerase mutants

[0064] The present application relates to a truncated form of a Bst DNA polymerase mutant, which is obtained by subjecting the original sequence of the Bst DNA polymerase to mutations and subsequent truncation. Specifically, the Bst DNA polymerase mutant is obtained by subjecting the original sequence of the Bst DNA polymerase to at least one mutation selected from the group consisting of K369G, A464G, and S619G. Preferably, the mutations K369G, A464G, and S619G are mutated, i.e., the K at position 369 is mutated to a G, the A at position 464 is mutated to a G, and the S at position 619 is mutated to a G, thereby obtaining the Bst DNA polymerase mutant. The Bst DNA polymerase mutant is then truncated to remove 1-290 amino acids at the N-terminus, thereby obtaining the truncated form of the Bst DNA polymerase mutant, wherein the specific amino acid sequence is selected from the group consisting of: (i) an amino acid sequence as shown in SEQ ID NO.2; and (ii) an amino acid sequence having greater than 95% homology to the sequence as shown in SEQ ID NO.2. It is understandable that the truncated version of the Bst DNA polymerase mutant as described above can be obtained by first truncating the original sequence of Bst DNA polymerase and then performing mutation in the same manner.

[0065] Polynucleotide sequence encoding the target gene

[0066] The present application also relates to a polynucleotide sequence encoding a target gene (encoding a Bst DNA polymerase mutant and a truncated form of the Bst DNA polymerase mutant).

[0067] In the present application, the problem of reduced yield when expressing heterologous proteins in Escherichia coli due to synonymous codon preference optimization is overcome. The present application relates to a polynucleotide sequence that has been optimized for synonymous codon preference. The obtained target gene sequence is optimized for synonymous codon preference. The target gene sequence that has been optimized for synonymous codon preference can express the same amino acid sequence as the target protein. In an embodiment of the present application, the polynucleotide sequence encoding the target gene is selected from any one of the following: (i) a polynucleotide with a sequence as shown in SEQ ID NO.1; (ii) a polynucleotide with a homology greater than 80% (more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%) to the sequence as shown in SEQ ID NO.1; and (iii) a polynucleotide complementary to the polynucleotide sequence described in (i) or (ii).

[0068] The nucleotide full-length sequence or its fragment of BstDNA polymerase mutant, the truncated form of BstDNA polymerase mutant or its element can be obtained by PCR amplification, recombination or artificial synthesis method usually.For PCR amplification, primers can be designed according to the disclosed relevant nucleotide sequence, especially the open reading frame sequence, and the cDNA library prepared by commercially available cDNA library or conventional method known to those skilled in the art is used as template to increase and obtain the relevant sequence.When the sequence is long, it is usually necessary to carry out two or more PCR amplifications, and then the fragments amplified each time are spliced ​​together in the correct order.In case the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is normally to clone it into a vector, then transfer it into cells, and then separate and obtain the relevant sequence from the host cell after the propagation by conventional methods.

[0069] In addition, the method for artificial synthesis can also be used to synthesize relevant sequences, especially when the fragment length is shorter. Usually, by synthesizing a plurality of small fragments first, and then connecting to obtain a very long fragment of sequence. The method for using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present application. The primer that is used for PCR can be suitably selected according to the sequence information of the present application disclosed herein, and can be synthesized by conventional method. Conventional method can be as separated and purified by gel electrophoresis amplification DNA / RNA fragment.

[0070] Expression vector containing the target gene

[0071] The present application also relates to vectors comprising the polynucleotides of the present application. 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 the host organism, another organism, a plasmid, or viral DNA, or may be synthetic. The vector may be synthetic or any expression vector readily amenable to recombinant DNA procedures, 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 application is an expression vector. In one embodiment of the present application, pET-28a(+) was selected as the vector to achieve more efficient expression efficiency.

[0072] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the present application protein and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The described DNA sequence can be effectively connected 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. Exemplarily, 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 sticky end connection of a single restriction enzyme site, the directional cloning of a double restriction enzyme fragment, the sticky end connection of different restriction enzyme sites, the blunt end connection, the artificial linker connection or the same oligonucleotide end connection can be used to realize the insertion of the exogenous DNA fragment.

[0073] Host cells containing the target gene

[0074] The application also relates to host cells produced by genetic engineering using the vectors or coding sequences of the application. A vector containing a codon-optimized target gene can be inserted, transfected, or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized target gene of the application and capable of expressing the target protein. In the application, 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, and the host cell is preferably a bacterium, and is preferably Escherichia coli, more preferably Escherichia coli Rosetta (DE3) strain.

[0075] Method for preparing target protein

[0076] The present application also relates to a method for preparing a target protein, which can be expressed or produced using the polynucleotide sequence of the present application. Generally speaking, the following steps are involved:

[0077] (1) transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding the protein of the present application, or with a recombinant expression vector containing the polynucleotide;

[0078] (2) host cells cultured in a suitable culture medium;

[0079] (3) Isolate and purify proteins from culture medium or cells.

[0080] 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.

[0081] The transformant obtained can be cultivated with a conventional method, and expresses the polypeptide encoded by the gene of the application. According to the host cell used, the substratum used in the cultivation can be selected from various conventional culture media, is preferably SB, TB, LB or SOC substratum. 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 target protein and promoting the expression of soluble protein, a preferred embodiment of the application uses the host cell of TB or LB substratum culture, and contains the kanamycin resistance gene in the substratum used. The output of using TB substratum supernatant to express target protein is slightly higher than LB substratum.

[0082] In order to further promote the soluble expression of the target protein, in a preferred embodiment of the present application, the host cells are cultured to an OD 600 After the pH value reaches 0.6-0.8, IPTG is used for induction, and the culture is continued for about 8-12 hours at 17-19° C. or 35-39° C. The soluble expression level is high at low temperature, such as 17-19° C.

[0083] The protein in the above method can be expressed in the cell or on the cell membrane or secreted outside the cell. If necessary, its physical, chemical and other characteristics can be utilized to separate and purify the protein by various separation methods. Therefore in the present application, after successfully cultivating and obtaining the target protein, it is also related to the step of separation and purification, for example, separation and purification of protein from the culture medium to obtain highly purified target protein. Although the method for purifying the target protein can be conventional means well known to those skilled in the art, including but not limited to: conventional renaturation treatment, treatment with protein precipitant (salting out method), centrifugation, infiltration breaking bacteria, 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 the combination of these methods. The preferred embodiment of the present application uses Ni column affinity chromatography and ion exchange method to carry out purification treatment to the expression product. In affinity chromatography, the solution composition used affects the chromatographic effect. In a preferred embodiment of the present application, the Buffer A solution used is composed as follows: Buffer A: 20mM sodium phosphate, 500mM NaCl, 20mM Imidazole, pH 7.4; Buffer B: 20mM sodium phosphate, 500mM NaCl, 500mM Imidazole, pH 7.4; and Buffer C: 20mM sodium phosphate, 1M NaCl, pH 7.4. In a preferred embodiment of the present application, a dialysis step is further included after Ni column affinity chromatography and ion exchange. The dialysate composition is as follows: 1x PBS, 10% Glycerol, pH 7.4.

[0084] In this application, any exemplary or exemplary wording (e.g., "") provided for certain embodiments herein is used only to better present this application and does not limit the scope of this application claimed in other ways. No wording herein should be interpreted as indicating an element not described in the claims that is indispensable for the implementation of this application.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] The present application also relates to variants of the aforementioned polynucleotides, which encode protein fragments, analogs, and derivatives having the same amino acid sequence as the present application. 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.

[0090] 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.).

[0091] 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.

[0092] 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.

[0093] 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.

[0094] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for the following examples that do not specify specific conditions 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.

[0095] 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.

[0096] Example 1

[0097] In this example, a plasmid encoding a target protein was prepared. The specific steps are as follows:

[0098] The full-length amino acid sequence of Geobacillus stearothermophilus Bst DNA polymerase provided by NCBI (WP_042379932.1) was used as a reference, as shown in SEQ ID NO: 3, the 1-290 amino acids at the N-terminus were truncated, and three amino acid sites (K / 369 / G, A / 464 / G, S / 619 / G) were mutated to form a truncated sequence (SEQ ID NO: 2). Combined with the experimental design requirements of this application, after optimization of the synonymous codon preference of Escherichia coli, the connection vector was pET-28a(+), and the C-terminal fusion expression (His)6 tag was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0099] SEQ ID NO:2

[0100] SEQ ID NO:3

[0101] >WP_042379932.1 DNA polymerase I

[0102] Example 2

[0103] In this example, the plasmid prepared in Example 1 was used to transform host cells to obtain host cells encoding the target protein. The specific steps are as follows:

[0104] Take 1 μL of the plasmid and transform it into competent E. coli BL21(DE3) via heat shock. Add antibiotic-free SOC medium and culture at 37°C with shaking for 50 minutes. Spread the bacterial solution evenly onto a kanamycin-resistant LB plate and incubate in a 37°C incubator overnight.

[0105] SEQ ID NO.1

[0106] Example 3

[0107] In this example, soluble expressed Bst DNA polymerase was prepared.

[0108] Single clones from Example 2 were selected and inoculated into TB and LB media, cultured with shaking at 37°C until the OD600 reached between 0.6 and 0.8. IPTG was then used for induction, and cultured with shaking overnight at 18°C. A control group without IPTG was used, and each experiment was repeated once. Samples were ultrasonically disrupted and analyzed by SDS-PAGE. The results are shown in Figure 1. The results show that under the induction conditions of 18°C, Bst DNA polymerase was soluble in the supernatant of both LB and TB media, with higher yields of the target protein in the TB supernatant compared to LB culture. The protein molecular weight was approximately 70 kDa, which is consistent with the protein size predicted on the Expasy website (70 kDa).

[0109] Example 4

[0110] In this example, the Bst DNA polymerase prepared in Example 3 was purified.

[0111] 1.5 L of bacterial culture was cultured in a shake flask with TB medium and the cells were collected by centrifugation. Approximately 18 g of cells were weighed and resuspended in lysis buffer on ice. The cells were disrupted by ultrasonication at low temperature and high speed for 30 min. The supernatant was collected and filtered through a membrane. The supernatant was passed through a Ni-column affinity chromatography and an ion exchange column, and 50 mL of the flow-through was collected and dialyzed overnight. The purified electrophoresis patterns are shown in Figures 2 and 3 below. The target protein concentration calculated by the BCA method was 2 mg / mL, the total protein amount was approximately 100 mg, and the yield was 5.56 mg / g of bacteria. The buffer preparation method during the affinity chromatography process is as follows:

[0112] Buffer A: 20 mM sodium phosphate, 500 mM NaCl, 20 mM Imidazole, pH 7.4

[0113] Buffer B: 20 ​​mM sodium phosphate, 500 mM NaCl, 500 mM Imidazole, pH 7.4

[0114] Buffer C: 20 mM sodium phosphate, 1 M NaCl, pH 7.4

[0115] Dialysate: 1x PBS, 10% Glycerol, pH 7.4.

[0116] Example 5

[0117] In this example, the activity of the Bst DNA polymerase purified in Example 4 was detected. The specific steps are as follows:

[0118] (1) Experimental materials

[0119] Sample: Bst DNA polymerase

[0120] Equipment and reagents: Real-time fluorescence quantitative PCR system, Picogreen, λ DNA (Merck).

[0121] Substrate T2:

[0122] The above substrate was synthesized, purified by HPLC, and prepared into a 100 pmol / μL mother solution according to experimental requirements.

[0123] Preparation of Bst enzyme dilution solution:

[0124] 10X PBS pH 7.4: 10 mL, glycerol: 10 mL. Mix the above materials and add ultrapure water to 100 mL. Filter once through a 0.22 μm membrane. Sterilize by high-temperature and high-pressure sterilization at 121°C for 30 min and store at -20°C.

[0125] (2) Experimental steps: 10× PCR Buffer is prepared as shown in Table 1 below:

[0126] Table 1

[0127] The λDNA pre-staining solution is prepared as shown in Table 2 below:

[0128] Table 2

[0129] λDNA was diluted in a gradient manner, with a total of 10 gradients, and the concentrations (ng / μL) from high to low were 100, 25, 20, 15, 10, 7.5, 5, 2.5, 1.25, and 0.625.

[0130] Dilution of test samples: Dilute the BST enzyme sample with BST enzyme diluent. Dilution multiples are 1 / 100, 1 / 200, 1 / 400, 1 / 800, 1 / 1600, and 1 / 3200. The dilution multiple can be adjusted according to actual conditions.

[0131] Reaction system configuration (single reaction system): Configuration as shown in Table 3 below:

[0132] Table 3. Polymerase systems tested

[0133] Mix the λDNA and λDNA pre-staining buffer at a 1:1 ratio for each dilution gradient. The 0 μg / mL λDNA group served as the control, while the NTC group added 1.3 μL of Bst DNA polymerase dilution. No sample enzyme was added to the λDNA group. After setting up the reaction program, place the 96-well plate in a qPCR instrument and monitor fluorescence in real time.

[0134] (3) Experimental results

[0135] Drawing of λDNA standard curve

[0136] The amount of λDNA input was used as the horizontal axis, and the net fluorescence value after deducting the average value of the NTC group was used as the vertical axis. A linear standard curve was drawn to make R2>0.99. The standard curve is shown in Figure 4:

[0137] Export the data from the instrument and select the fluorescence values ​​at cycle 25 for each Bst DNA polymerase gradient and NTC group. Calculate the average value for each Bst DNA polymerase gradient and subtract the average value for the NTC group to obtain the net fluorescence value. Substitute the net fluorescence value into the λDNA standard curve to calculate the amount of DNA generated, A1. A1 / 649 is used to calculate the amount of dNTPs consumed in the reaction, A2, in nmol. Finally, calculate the Bst DNA polymerase activity using the formula (A2 * dilution factor) / 1.29.

[0138] Enzyme activity definition: Using a synthetic hairpin oligonucleotide sequence as a template, the amount of enzyme required to incorporate 1.29 nmol dNTPs in 1 minute at 65°C is defined as 1 U. The calculation results are shown in Table 4 below:

[0139] Table 4

[0140] Results: λDNA concentration standard curve R 2 >0.99, and the activity of the self-produced Bst DNA polymerase was 514 U / μL. According to inter-batch enzyme testing results, when the CV value was within 10% and the dNTPs consumption was between 0.03 and 0.25 nmol, the activity of Bst DNA polymerase was similar across the gradient intervals.

[0141] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present application, 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 application.

Claims

1. A truncated form of a Bst DNA polymerase mutant, characterized in that: The amino acid sequence of the truncated form of the Bst DNA polymerase mutant is the amino acid sequence shown in SEQ ID NO.

2.

2. An isolated polynucleotide encoding a truncated form of a Bst DNA polymerase mutant, characterized in that The polynucleotide is codon-optimized, and the polynucleotide is the polynucleotide shown in SEQ ID NO.

1.

3. An expression vector, characterized in that: The expression vector comprises the polynucleotide according to claim 2.

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

5. A host cell, characterized in that The host cell comprises the expression vector according to claim 3 or 4; or The polynucleotide according to claim 2 is integrated into the genome of the host cell.

6. A method for preparing a truncated form of a Bst DNA polymerase mutant, characterized in that: The method comprises the steps of: Transforming a host cell with the polynucleotide vector as claimed in claim 2; The host cell is cultured to express the truncated form of the Bst DNA polymerase mutant.

7. The method according to claim 6, characterized in that Using TB medium or LB medium to culture the host cells; and / or, culturing the host cell at a temperature of 16 to 19° C.; And / or, when culturing the host cell, the culture medium used contains a kanamycin resistance gene.

8. The method according to claim 6, characterized in that When the host cells are cultured, they are induced by IPTG to express the target protein.

9. The method according to claim 8, characterized in that The method further comprises the step of separating the target protein, wherein the step of separating the target protein comprises eluting the crushed target protein supernatant with a mobile phase through a chromatography column at the same time, and collecting the eluate.

10. A kit, characterized in that: The kit comprises: a truncated form of the Bst DNA polymerase mutant according to claim 1; or The polynucleotide of claim 2; or The expression vector according to claim 3 or 4; or The host cell according to claim 5.

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