WO2025092028A1 - Mutant bst dna polymerase large fragment, and preparation method therefor - Google Patents

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

Priority declaration

This application claims priority to Chinese patent application No. 2023114455020, filed on November 2, 2023. This application cites the entire text of the above patent application.

Technical Field

The present application relates to the field of biomedicine, and in particular to truncated forms of Bst DNA polymerase mutants, and preparation methods and applications thereof.

Background Art

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus. The gene is 2631bp long, the amino acid sequence is 876aa long, the protein molecular weight is 99kDa, and the isoelectric point is 5.32. The complete Bst DNA polymerase has four activities: 5'-3' exonuclease activity, 5'-3' DNA polymerase activity, 3'-5' exonuclease activity (correction activity), and strand displacement activity. The C-terminal peptide chain is composed of amino acids 291-876, and performs the other three enzyme activities except 5'-3' exonuclease activity, which is called Bst DNA polymerase large fragment. The optimal reaction temperature of Bst DNA polymerase is 65℃. It is inactivated at a temperature above 80℃, so it cannot be used for thermal cycle sequencing or PCR. It has strong stress resistance, good thermal stability, and high tolerance to non-ionic surfactants and high salt.

Based on the above characteristics of Bst DNA polymerase, the enzyme is used in DNA sequencing rich in GC base pairs, rapid sequencing of low-content template DNA, isothermal amplification of DNA, multiple strand displacement amplification and whole genome amplification. In addition, Bst DNA polymerase can also initiate template-dependent DNA synthesis and randomly add nucleotides at the 3' end. In recent years, nucleic acid isothermal amplification technology has developed rapidly, mainly including rolling circle amplification (RCA) technology and loop-mediated isothermal amplification (LAMP) technology. Bst DNA polymerase is the basic enzyme for these two technologies. Since Bst DNA polymerase can perform amplification reactions under constant temperature conditions, ordinary water baths or equipment with stable heat sources can meet the reaction requirements. Compared with conventional PCR detection, it does not rely on expensive PCR instruments and professional laboratories, and the detection time is short (only 1/3-1/5 of PCR technology). It has obvious advantages, so it is widely used in various fields such as medical testing, inspection and quarantine of pathogenic microorganisms, 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 cyclic heating and cooling during library amplification, improves amplification efficiency, and reduces sequencing time. Existing sequencing platforms using Bst DNA polymerase include Roche, Illumina, Solexa, etc.

Although Bst DNA polymerase has been commercialized, the quality of Bst DNA polymerase in the domestic market is uneven compared to foreign commercial enzymes, and the localization process is slow. It is mainly purchased from various foreign biological companies. Although this method is simple and quick to operate, it is expensive. If it is possible to construct an engineered bacterium that expresses the Bst DNA polymerase gene and synthesize Bst DNA polymerase by itself, the cost of scientific research and production can be greatly reduced, and it is also conducive to the localization of Bst DNA polymerase. In the prior art, the method for preparing Bst DNA polymerase cannot be expressed in the supernatant, the purification cost is high, and the product activity is poor. Therefore, this field urgently needs to develop a low-cost method for preparing Bst DNA polymerase that is conducive to the expression of supernatant products with high activity.

Summary of the invention

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

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

Another object of the present application is to provide a polynucleotide sequence encoding a truncated version of the Bst DNA polymerase mutant.

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

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

In order 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:

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

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

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 the polynucleotide is selected from any one of the following:

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

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

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

In a third aspect of the present application, an expression vector is provided, wherein the expression vector comprises the polynucleotide provided in the second aspect of the present application.

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

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

The polynucleotide provided in the second aspect of the present application is integrated into the genome of the host cell.

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

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

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

Separating the target protein to obtain a truncated form of the Bst DNA polymerase mutant;

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

In some preferred embodiments, the step of separating the target protein further comprises:

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

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

The fifth aspect of the present application provides a kit, which includes: a truncated form of the Bst DNA polymerase mutant provided in the first aspect of the present application; or

A polynucleotide as provided in the second aspect of the present application; or

As the expression vector provided in the third aspect of the present application; or

A host cell as described in the fourth aspect of the present application.

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

(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 E. coli system;

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

It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

Figure 1 is an SDS-PAGE identification diagram of the truncated product of the Bst DNA polymerase mutant according to the embodiment of the present application;

Figure 2 is a graph showing the results of nickel column purification of the truncated Bst DNA polymerase mutant according to the example of the present application;

Figure 3 is a diagram of the ion exchange purification of the truncated Bst DNA polymerase mutant according to the example of the present application;

Figure 4 is a standard curve diagram of the enzyme activity test of the truncated form of the Bst DNA polymerase mutant in the example of the present application.

DETAILED DESCRIPTION

In the prior art, the prepared Bst DNA polymerase has poor activity, low yield and poor activity. After extensive and in-depth research, the applicant obtained its mutants and truncated mutants by mutating and truncating Bst DNA polymerase, and then analyzed the mutated and/or truncated Bst DNA polymerase to obtain the target gene sequence encoding it, optimized the target gene sequence to make it suitable for the vector and host cell expression system of the present application, and successfully expressed Bst DNA polymerase with good enzyme activity.

Bst DNA polymerase mutant

The present application relates to a Bst DNA polymerase mutant, which is obtained by mutation at at least one site selected from the following based on the original sequence of Bst DNA polymerase: K369, A464 and S619. Further, it is obtained by mutation at three sites, K369, A464 and S619, based on the original sequence of Bst DNA polymerase. The mutation here can be substitution, addition or deletion of amino acids.

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. Furthermore, the Bst DNA polymerase mutant undergoes K369G, A464G and S619G mutations based on the original sequence of the Bst DNA polymerase, that is, the K at position 369 is mutated to G, the A at position 464 is mutated to G, and the S at position 619 is mutated to G, thereby obtaining the Bst DNA polymerase mutant. The aforementioned mutants can be obtained by conventional in vitro site-directed mutagenesis methods in the art, for example, by introducing the desired changes into the target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR), including the addition, deletion, and point mutation of bases.

Bst DNA polymerase mutant truncations

The present application relates to a truncated form of a Bst DNA polymerase mutant, which is obtained by mutation based on the original sequence of the Bst DNA polymerase and then truncation. Specifically, the Bst DNA polymerase mutant is obtained by first undergoing at least one mutation selected from the following on the basis of the original sequence of the Bst DNA polymerase: K369G, A464G and S619G; preferably, mutations of K369G, A464G and S619G occur, i.e., mutation of K at position 369 to G, mutation of A at position 464 to G, and mutation of S at position 619 to G, to obtain a Bst DNA polymerase mutant. Then, the Bst DNA polymerase mutant is truncated, and the 1-290 amino acids at the N-terminus are cut off to obtain a truncated form of the Bst DNA polymerase mutant, wherein the specific amino acid sequence is selected from any one of the following: (i) an amino acid sequence as shown in SEQ ID NO.2; and (ii) an amino acid sequence having a homology greater than 95% with the sequence as shown in SEQ ID NO.2. It can be understood that the truncated form of the Bst DNA polymerase mutant as described above can also be obtained by first truncating the original sequence of the Bst DNA polymerase and then mutating it in the same manner.

Polynucleotide sequence encoding the target gene

The present application also relates to a polynucleotide sequence encoding a target gene (encoding a Bst DNA polymerase mutant and a truncated version of a Bst DNA polymerase mutant).

In the present application, the problem of reduced yield when expressing heterologous proteins in Escherichia coli by synonymous codon preference optimization is overcome, and the present application relates to a polynucleotide sequence optimized by synonymous codon preference. The obtained target gene sequence is subjected to synonymous codon preference optimization, and the target gene sequence optimized by 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 of the sequence shown in SEQ ID NO.1; (ii) a polynucleotide having a homology greater than 80% (more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%) with the sequence shown in SEQ ID NO.1; and (iii) a polynucleotide complementary to the polynucleotide sequence described in (i) or (ii).

In the present application, the full-length nucleotide sequence or fragments of the Bst DNA polymerase mutant, the truncated form of the Bst DNA polymerase mutant or its elements can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the relevant nucleotide sequences that have been disclosed, 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 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 in each time together in the correct order. Once the relevant sequence is obtained, the recombinant method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.

In addition, the method for artificial synthesis can also be used to synthesize the relevant sequence, 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 the sequence. The method for using the PCR technology to amplify DNA/RNA is preferably used to obtain the gene of the present application. The primer used for PCR can be appropriately selected according to the sequence information of the present application disclosed herein, and can be synthesized by conventional methods. Conventional methods can be used such as separation and purification of amplified DNA/RNA fragments by gel electrophoresis.

Expression vector containing the target gene

The present application also relates to a vector comprising a polynucleotide of the present application. In the present application, "vector" means a linear or circular DNA molecule comprising a fragment encoding a target protein, which is operably connected to other fragments providing its transcription. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selectable markers, enhancers, polyadenylation signals, vectors, etc. The vector fragment may be derived from a host organism, another organism, a plasmid or viral DNA, or may be synthetic. The vector may be any expression vector that is synthetic or conveniently performs recombinant DNA manipulation, and the selection of the vector generally depends on the host cell into which the vector is to be introduced. Therefore, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, and the replication of the extrachromosomal entity is unrelated to chromosomal replication, such as a plasmid. Alternatively, the vector may be a vector that is integrated into the host cell genome and replicated together with the chromosome into which it is integrated when introduced into the host cell. In one embodiment, the vector of the present application is an expression vector. pET-28a (+) is selected as a vector in an embodiment of the present application to obtain a more efficient expression efficiency.

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 application and suitable transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to a suitable 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 carrier DNA molecule into a linear molecule that can be connected to an exogenous gene, and then the codon-optimized target gene fragment is connected to the carrier, and 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 flat end connection, the artificial joint connection or the same oligonucleotide end connection can be used to realize the insertion of the exogenous DNA fragment.

Host cells containing the target gene

The present application also relates to a host cell produced by genetic engineering using the vector or coding sequence of the present application. The vector containing the codon-optimized target gene can be inserted, transfected or otherwise transformed into a host cell by a known method, thereby obtaining a transformant containing the codon-optimized target gene of the present application and capable of expressing the target protein. In the present application, a "host cell" is a cell into which an exogenous polynucleotide and/or vector is introduced. The host cell can be a eukaryotic host cell or a prokaryotic host cell, and the host cell is preferably a bacterium, and preferably Escherichia coli, more preferably Escherichia coli ROSETTA (DE3) strain.

Method for preparing target protein

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:

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

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

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

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.

The transformant obtained can be cultivated by conventional methods to express the polypeptide encoded by the gene of the present application. According to the host cell used, the culture medium used in the cultivation can be selected from various conventional culture media, preferably SB, TB, LB or SOC culture medium. Cultivate under conditions suitable for host cell growth. After the host cell grows to a suitable cell density, induce the promoter selected by 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 improving the expression of soluble protein, a preferred embodiment of the present application uses the host cell cultivated with TB or LB culture medium, and contains the kanamycin resistance gene in the culture medium used. The output of using the TB culture medium supernatant to express the target protein is slightly higher than the LB culture medium.

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 between 0.6 and 0.8, induced with IPTG, and cultured for about 8 to 12 hours at 17 to 19° C. or 35 to 39° C. The soluble expression level is high at a low temperature range, such as 17 to 19° C.

The protein in the above method can be expressed in the cell or 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 characteristics. Therefore, in the present application, after successfully culturing to obtain the target protein, it is also related to the step of separating and purifying it, for example, separating and purifying the protein from the culture medium to obtain a highly purified target protein. Although the method for purifying the target protein is conventional means well known to those skilled in the art, including but 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 the combination of these methods. In a preferred embodiment of the present application, Ni column affinity chromatography and ion exchange method are used to purify the expression product. In affinity chromatography, the solution components used affect the chromatography effect. In the preferred embodiment of the present application, the Buffer A solution used is composed of: 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. The preferred embodiment of the present application also includes a dialysis step after Ni column affinity chromatography and ion exchange method, and the dialysate is composed of: 1x PBS, 10% Glycerol, pH 7.4.

In this application, any exemplary or exemplary wording (e.g., "") provided for certain embodiments herein is used only to better present the present application, and does not limit the scope of the present application 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 application.

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

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

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) that is present in the nucleic acid or polypeptide's 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 sources.

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 artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

The present application also relates to variants of the above-mentioned polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present application. The variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the encoded polypeptide.

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

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 the polynucleotide or polypeptide is arranged, and the number of positions containing the same nucleotide or amino acid residue 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. The polynucleotide can be different in one position, for example, according to the presence of different nucleotides (i.e., substitutions or variations) or nucleotide deletions (i.e., insertion or deletion of one or two nucleotides in the polynucleotide). The polypeptide can be different in one position, for example, by containing an amino acid (i.e., substitutions or variations) or an amino acid deletion (i.e., insertion of an amino acid or amino acid deletion in one or two polypeptides). The 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 nucleotides or amino acid residues in the polynucleotide or polypeptide and then multiplying by 100.

As used herein, the terms "sequence complement" and "reverse sequence complement" are used interchangeably and 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, its reverse complementary sequence is GTTCAT.

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 cell or expression product may be harvested, i.e., recovered.

To make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the present application is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually based on normal conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

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 the 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 the present application.

Example 1

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

Using the full-length amino acid sequence of Bacillus stearothermophilus Bst DNA polymerase provided by NCBI (WP_042379932.1) 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.

SEQ ID NO:2

SEQ ID NO:3

>WP_042379932.1 DNA polymerase I

Example 2

In this example, the plasmid prepared in Example 1 is used to transform host cells to obtain host cells containing the encoded target protein. The specific steps are as follows:

Take 1 μL of plasmid and transform it into competent E. coli BL21 (DE3) by heat shock method, add SOC medium without antibiotics, and culture at 37℃ with shaking for 50 minutes. Spread the bacterial solution evenly on LB plate containing kanamycin resistance and culture in 37℃ incubator overnight.

SEQ ID NO.1

Example 3

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

The monoclone in Example 2 was picked up and inoculated into TB and LB culture medium. The culture was shaken at 37°C until OD600 was between 0.6 and 0.8. IPTG was induced and the culture was shaken at 18°C overnight. The group without IPTG was used as a control. Each group of experiments was repeated once. The samples were ultrasonically broken for SDS-PAGE identification. The results are shown in Figure 1. The results showed that under the induction condition of 18°C, Bst DNA polymerase could be expressed soluble in the supernatant in both LB and TB culture medium, and the yield of the target protein in the supernatant of TB culture medium was higher than that in LB culture medium. The protein molecular weight was about 70Kda, which was basically consistent with the protein size (70Kda) predicted on the Expasy website.

Example 4

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

TB medium shake flask culture 1.5L bacterial liquid, centrifuge to collect bacteria. Weigh about 18g of bacteria, add lysis solution and resuspend on ice, ultrasonically disrupt cells, low temperature and high speed for 30min, take the supernatant, filter the supernatant through Ni-column affinity chromatography and ion exchange column, collect 50mL of penetration, dialyze overnight, and the purified electrophoresis diagram is shown in Figures 2 and 3 below. The concentration of the target protein calculated by the BCA method is 2mg/mL, the total protein amount is about 100mg, and the yield is 5.56mg/g bacteria. The buffer preparation method during affinity chromatography is as follows:

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

Buffer B: 20 mM sodium phosphate, 500 mM NaCl, 500 mM Imidazole, pH 7.4

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

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

Example 5

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

(1) Experimental Materials

Sample: Bst DNA polymerase

Equipment and reagents: Real-time fluorescence quantitative PCR system, Picogreen, λDNA (Merck).

Substrate T2:

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

Preparation of Bst enzyme dilution:

10X PBS pH 7.4: 10mL, glycerol: 10mL, mix the above materials, add ultrapure water to 100mL, filter once with 0.22μm membrane, sterilize at 121℃ and high temperature and high pressure for 30min, and store at -20℃.

(2) Experimental steps 10×PCR Buffer is configured as shown in Table 1:

Table 1

λDNA pre-staining solution is prepared as shown in Table 2 below:

Table 2

The 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, respectively.

Dilution of the sample to be tested: Bst enzyme sample is diluted with Bst enzyme diluent. The dilution multiple is 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200. The dilution multiple can be adjusted according to the actual situation.

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

Table 3. Polymerase systems to be tested

Each dilution gradient of λDNA and λDNA pre-staining solution was mixed evenly at a ratio of 1:1. The 0ug/mlλDNA group was used as the control group, and 1.3μL Bst DNA polymerase dilution solution was added to the NTC group. No sample enzyme was added to the λDNA group. After setting up the reaction program, the 96-well plate was placed in the qPCR instrument to monitor the fluorescence value in real time.

(3) Experimental results

Plotting of λDNA standard curve

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:

Export the data from the computer, select the fluorescence values of each gradient Bst DNA polymerase experimental group and the experimental group NTC group at the 25th cycle, calculate the average value of each gradient Bst DNA polymerase, subtract the average value of the NTC group, and get 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 obtain the amount of dNTPs consumed in the reaction A2, in nmol. Finally, the activity of Bst DNA polymerase is calculated using the formula (A2*dilution factor)/1.29.

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

Table 4

Results: The standard curve of λDNA concentration R ² > 0.99, the activity of self-produced Bst DNA polymerase was 514U/μL. According to the results of the batch enzyme test, when the CV value was within 10% and the amount of dNTPs consumed was 0.03-0.25nnmol, the activity results of Bst DNA polymerase in each gradient interval were close.

Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present application, and 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 (10)

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. A separated polynucleotide encoding a truncated form of a Bst DNA polymerase mutant, characterized in that the polynucleotide is codon optimized and is a polynucleotide as shown in SEQ ID NO.1. An expression vector, characterized in that the expression vector comprises the polynucleotide according to claim 2. The expression vector according to claim 3 is characterized in that the expression vector is an Escherichia coli expression vector. 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. 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 a truncated form of the Bst DNA polymerase mutant. The method according to claim 6, characterized in that the host cell is cultured using TB medium or LB medium; 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. The method according to claim 6, characterized in that when culturing the host cells, IPTG is induced to express the target protein. The method according to claim 8 is 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 through a chromatography column simultaneously with the mobile phase and collecting the eluate. A kit, characterized in that the kit comprises: a truncated form of the Bst DNA polymerase mutant as described in 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.