Mutant pwt polymerase and methods of making and using same

Abstract

The application discloses a mutant PWT polymerase and a preparation method and application thereof, and relates to the technical field of biology. The mutant PWT polymerase has any one of A1) to A2): A1), an amino acid sequence shown in amino acids 407 to 986 of SEQ ID NO. 1; A2), an amino acid sequence obtained by connecting a tag to the N terminal or / and C terminal of the amino acid sequence shown in A1). The mutant PWT polymerase can obviously perform isothermal amplification under room temperature conditions and can tolerate a higher salt concentration, and is expected to become an ideal tool for rolling circle amplification and multiple displacement amplification.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to mutant PWT polymerase, its preparation method, and its application. Background Technology

[0002] DNA polymerases are responsible for genome replication and maintenance, a crucial function for the accurate transmission of genetic information from generation to generation. Strand substitution refers to the ability of polymerases to replace, rather than degrade, downstream DNA encountered during DNA synthesis. During strand substitution replication, only one DNA strand is replicated at a time. Strand substitution synthesis releases single-stranded DNA, which is then replicated into double-stranded DNA. The most commonly used polymerase capable of isothermal amplification at room temperature is phi29 polymerase, but its exonuclease activity limits its application in nanopore sequencing. PWT polymerase, a DNA polymerase derived from the psychrophilic bacterium BL-248-WT-3, can be used for sequencing, but it lacks significant strand substitution ability, exhibiting high activity only at low temperatures. It cannot perform efficient isothermal amplification at room temperature and is intolerant of high salt concentrations. Therefore, continuous improvement of the properties of PWT polymerase is necessary. Summary of the Invention

[0003] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a mutant PWT polymerase that can perform significant isothermal amplification at room temperature and tolerate high salt concentrations, making it a promising tool for rolling circle amplification and multiple substitution amplification.

[0004] The present invention also provides biological materials related to the above-mentioned mutant PWT polymerase.

[0005] The present invention also provides an enzyme preparation.

[0006] The present invention also provides a method for preparing the above-mentioned mutant PWT polymerase.

[0007] The present invention also provides a method for replicating, amplifying, or sequencing template DNA.

[0008] The present invention also provides applications of the above-mentioned mutant PWT polymerase, biomaterials or enzyme preparations.

[0009] According to a first aspect of the present invention, a mutant PWT polymerase has any one of A1) to A2):

[0010] A1) The amino acid sequence shown in amino acids 407 to 986 of SEQ ID NO.1;

[0011] A2) is an amino acid sequence obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in A1) or A2).

[0012] The mutant PWT polymerase according to embodiments of the present invention has at least the following beneficial effects:

[0013] The mutant PWT polymerase in this embodiment can perform isothermal amplification at room temperature and can tolerate high salt concentrations, making it a promising tool for rolling circle amplification and multiple substitution amplification, with excellent application prospects in nanopore sequencing and other fields.

[0014] According to some embodiments of the present invention, the mutant PWT polymerase may also have an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in A1), and the function is the same or similar to that of the amino acid sequence shown in A1).

[0015] According to some embodiments of the present invention, the plurality is 2 to 100.

[0016] According to some embodiments of the present invention, the mutant PWT polymerase has an amino acid sequence as shown in SEQ ID NO.1.

[0017] According to some embodiments of the present invention, the amino acid sequence shown in A2) has at least 90% identity with the amino acid sequence of SEQ ID NO.1. Preferably, it is 95%.

[0018] According to some embodiments of the present invention, the tag includes at least one of the tags that facilitate the dissolution, purification, and detection of mutant PWT polymerase. It is understood that the mutant PWT polymerase of the present invention may contain one or more tags; multiple tags may comprise a combination of multiple identical tags or a combination of multiple different tags. For example: tags facilitating the dissolution of mutant PWT polymerase may be nus tags, maltose-binding proteins, or N-utilization substance A proteins; tags facilitating the purification of mutant PWT polymerase may be strep tags, His tags, GST tags, pelB signal sequences, ompA signal sequences, enterotoxin B-subunit signal sequences, alkaline phosphatase signal sequences, or FLAG octapeptides; tags facilitating the detection of mutant PWT polymerase may be horseradish peroxidase (HRP), β-galactosidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or blue fluorescent protein (BFP).

[0019] According to some embodiments of the present invention, the tag may specifically be a maltodextrin and / or a strep tag.

[0020] According to some embodiments of the present invention, a linker is further included between the tag and the mutant PWT polymerase.

[0021] According to a second aspect of the present invention, the biomaterial is related to the mutant PWT polymerase described in the first aspect of the present invention, wherein the biomaterial is any one of B1) to B4):

[0022] B1) A nucleic acid molecule encoding the mutant PWT polymerase described in the first aspect of the present invention;

[0023] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0024] B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0025] B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0026] According to some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as nucleotides 1219 to 2958 of SEQ ID NO.2.

[0027] According to some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule may also be as shown in SEQ ID NO.2.

[0028] According to some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the mutant PWT polymerase in a host cell. This DNA may include not only a promoter for initiating transcription of the mutant PWT polymerase gene, but also a terminator for terminating transcription of the protein gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0029] According to some embodiments of the present invention, the vector may be a plasmid, a granule, a bacteriophage, or a viral vector. Specifically, it may be the PET-21a vector.

[0030] According to some embodiments of the present invention, the recombinant vector may be a recombinant vector obtained by inserting a nucleic acid molecule encoding the mutant PWT polymerase into the multiple cloning site of the vector.

[0031] According to some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells, or insect cells. The bacteria may be *Escherichia coli*, such as *Escherichia coli* BL21(DE3). The recombinant organism does not contain reproductive material.

[0032] According to some embodiments of the present invention, the recombinant biological cell is a recombinant biological cell obtained by introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into a biological cell. Specifically, it can be recombinant Escherichia coli obtained by introducing the recombinant vector into Escherichia coli BL21(DE3).

[0033] An enzyme preparation according to a third aspect of the present invention includes the mutant PWT polymerase described in the first aspect of the present invention.

[0034] According to some embodiments of the present invention, the enzyme preparation can be used for in vitro DNA synthesis, DNA amplification, or DNA sequencing.

[0035] According to some embodiments of the present invention, the enzyme preparation further includes at least one of nucleoside triphosphate and reaction buffer. It is understood that the reaction buffer does not affect the activity of the mutant PWT polymerase.

[0036] The method for preparing the mutant PWT polymerase according to the first aspect of the present invention, as described in the fourth aspect embodiment of the present invention, includes:

[0037] The coding gene of the mutant PWT polymerase described in the first aspect of the present invention is introduced into a biological cell to express the coding gene, thereby obtaining the mutant PWT polymerase.

[0038] According to some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells.

[0039] According to some embodiments of the present invention, the prokaryotic cells include bacteria or algae. The bacteria may be *Escherichia coli*, such as *Escherichia coli* BL21(DE3).

[0040] According to some embodiments of the present invention, the eukaryotic cells include fungal, mammalian, or insect cells.

[0041] A method for replicating, amplifying, or sequencing template DNA according to a fifth aspect embodiment of the present invention includes:

[0042] The mutant PWT polymerase described in the first aspect of the present invention is mixed with template DNA and reaction reagents.

[0043] According to some embodiments of the present invention, the reaction reagents include a reaction buffer, primers, and nucleoside triphosphates. They may also include BSA and salts.

[0044] According to some embodiments of the present invention, the salt concentration of the reaction reagent is 0 to 300 mM.

[0045] According to some embodiments of the present invention, the reaction temperature after mixing is 25°C to 40°C.

[0046] According to the sixth aspect of the present invention, any one of C1) to C3) is applied in any one of D1) to D4).

[0047] C1) The mutant PWT polymerase described in the first aspect of the present invention;

[0048] C2), the biomaterials described in the second aspect of the present invention;

[0049] C3), the enzyme preparation described in the third aspect of the present invention;

[0050] D1) Nucleic acid amplification;

[0051] D2) Preparation of nucleic acid amplification-related products;

[0052] D3), ​​sequencing;

[0053] D4) Prepare sequencing-related products.

[0054] According to some embodiments of the present invention, the nucleic acid amplification includes strand displacement reaction, polymerase chain reaction, and isothermal amplification reaction. The isothermal amplification reaction is selected from loop-mediated amplification (LAMP), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA), and cross-primer amplification (CPA).

[0055] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0056] Figure 1 The results of SDS-PAGE protein electrophoresis of the fusion polymerase and wild-type PWT polymerase of the present invention are shown. Lanes 1 to 5 contain 160 ng, 320 ng, 480 ng, 640 ng, and 800 ng of bovine serum albumin (BSA, protein size 66 kDa), respectively. Lane 6 contains the fusion polymerase, and lane 7 contains the wild-type PWT polymerase.

[0057] Figure 2 This is the result of polymerase activity detection under different salt concentrations according to an embodiment of the present invention; wherein, lanes 1 to 3 refer to the rolling circle replication activity of wild-type PWT polymerase under salt concentrations of 0 mM, 150 mM and 300 mM, respectively, and lanes 4 to 6 refer to the rolling circle replication activity of fusion polymerase under salt concentrations of 0 mM, 150 mM and 300 mM, respectively.

[0058] Figure 3This is the result of polymerase activity detection at different temperatures according to an embodiment of the present invention; wherein, lane 1 is a negative control, with no enzyme added, and lanes 2 to 6 are the reaction products at 10℃, 25℃, 30℃, 35℃, and 40℃, respectively. Detailed Implementation

[0059] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0060] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0061] In the description of this invention, it should be noted that, unless otherwise explicitly defined, terms such as "setting," "installation," and "connection" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0062] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0063] Unless otherwise specified, "about" in this invention means that the allowable error is within ±2%.

[0064] In the following examples, the formulation of the 10× reaction buffer is: 500mM Tris-HCl, 100mM MgCl2, 100mM (NH4)2SO4 (pH 7.5).

[0065] Unless otherwise defined, all scientific or technical terms used in this patent are consistent with the common understanding of most people of ordinary skill in the art.

[0066] The term "nucleotide" generally refers to a compound formed by a nucleoside linked to an acidic molecule or group via an ester bond. For example, a nucleoside phosphate ester typically has one, two, or three phosphate groups covalently attached to the 5-position of the nucleoside's sugar group. In some cases, the definition of a nucleotide also includes homologues or analogues of typical nucleotides.

[0067] The term "amino acid" refers to the basic building blocks of proteins, giving them a specific molecular structure and enabling them to possess biochemical activity. For example, the "amino acids" used in this invention include the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0068] The term "amplification" refers to the process by which the number of target nucleic acid fragments increases under the action of nucleic acid polymerase.

[0069] Example 1 (Expression and purification of mutant PWT polymerase)

[0070] The expression sequence of the fusion polymerase was ligated into the gene expression vector pET-21a and transformed into *E. coli* competent cells BL21 (Shanghai Tulugang Biotechnology Co., Ltd., catalog number CC96107). After resistance selection, 5 μL of the bacterial culture was inoculated into 5 mL of LB broth and incubated overnight at 37°C with shaking. Then, it was transferred to 200 mL of LB broth and incubated at 37°C until OD = 1. IPTG was then added to a final IPTG concentration of 0.6 mM, and incubation continued at 20°C for 24 h. The bacterial cells were collected, sonicated, and the supernatant was used for Strep- The supernatant was purified by affinity chromatography using an XT 4F flow column (IBA-lifesciences, catalog number: 2-5010-010) to obtain the fusion polymerase, which was then concentrated using an AMICON ULTRA 15 mL 50K ultrafiltration column (Millipore, catalog number: UFC905024).

[0071] The amino acid sequence of the fusion polymerase is shown in SEQ ID NO.1.

[0072]

[0073] In this diagram, only the bolded portion represents the amino acid sequence of the maltose-binding protein fused with the mutant PWT polymerase, the italicized portion represents the linker, the bolded and underlined portion represents the amino acid sequence of the purification tag (strep tag), and the underlined portion represents the amino acid sequence of the mutant PWT polymerase.

[0074] The nucleotide sequence encoding the fusion polymerase is shown in SEQ ID NO.2.

[0075]

[0076]

[0077]

[0078] In this diagram, only the bolded portion represents the nucleotide sequence encoding maltose-binding protein, the italicized portion represents the linker encoding sequence, the bolded and underlined portion represents the nucleotide sequence encoding the purification tag (strep tag), and the underlined portion represents the nucleotide sequence encoding the mutant PWT polymerase.

[0079] Comparative Example 1 (Expression and purification of wild-type PWT polymerase)

[0080] The expression sequence of wild-type PWT polymerase was ligated into the gene expression vector pET-21a and transformed into *E. coli* competent cells BL21 (Shanghai Tulugang Biotechnology Co., Ltd., catalog number CC96107). After resistance selection, 5 μL of the bacterial culture was inoculated into 5 mL of LB broth and incubated overnight at 37°C. Then, it was transferred to 200 mL of LB broth and incubated at 37°C until OD = 1. IPTG was then added to a final concentration of 0.6 mM, and the culture was continued at 20°C for 24 h. The bacterial cells were collected, sonicated, and the supernatant was collected and purified using Nitrogen peroxide (NiO2). 2+ The supernatant was purified by affinity chromatography to obtain wild-type PWT polymerase, which was then concentrated using an AMICON ULTRA 15 mL 50K ultrafiltration column (Millipore, catalog number: UFC905024).

[0081] The amino acid sequence of the wild-type PWT polymerase is shown in SEQ ID NO.3.

[0082]

[0083]

[0084] The bolded and underlined portion represents the amino acid sequence of the purification tag (His tag).

[0085] The nucleotide sequence encoding the fusion polymerase is shown in SEQ ID NO.4.

[0086]

[0087]

[0088] The bolded and underlined portion represents the nucleotide sequence encoding the purification tag (His tag).

[0089] Detection Example 1

[0090] This test example validated the protein size and purity of the fusion polymerase of Example 1 and the wild-type PWT polymerase of Comparative Example 2 by SDS-PAGE protein electrophoresis.

[0091] The results are as follows Figure 1 As shown.

[0092] The protein size of the fusion polymerase is approximately 110 kDa, while the protein size of the wild-type PWT polymerase is similar to that of BSA; and both the purified fusion polymerase and wild-type PWT polymerase have high purity.

[0093] Detection Example 2

[0094] This test example assesses the rolling circle replication activity of the fusion polymerase of Example 1 and the wild-type PWT polymerase of Comparative Example 2 at different salt concentrations. The detection method is as follows:

[0095] The reaction system shown in Table 1 was reacted at 30°C for 3 hours, and the reaction product was then subjected to electrophoresis.

[0096] Table 1

[0097] reagents Final concentration BSA 0.5 μg / μL 10× reaction buffer 1× Template (M13mp18) 5nM Primers 5nM dNTP (10mM) 200nM KCl 0mM / 150mM / 300mM Fusion polymerase of Example 1 / Wild-type PWT polymerase of Comparative Example 2 200nM

[0098] The nucleotide sequence of the primer is 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.

[0099] Electrophoresis results as follows Figure 2 As shown.

[0100] The fusion polymerase exhibits strong chain substitution ability at 30°C and under high salt conditions of 300 mM, while the wild-type PWT polymerase does not have chain substitution ability at 30°C and cannot perform rolling circle replication.

[0101] Detection Example 3

[0102] This test example measures the rolling circle replication activity of the fusion polymerase from Example 1 at different temperatures. The detection method is as follows:

[0103] The reaction system shown in Table 1 (KCl concentration of 0 mM) was reacted at 10℃, 25℃, 30℃, 35℃ and 40℃ for 3 h, and the reaction product was then subjected to electrophoresis.

[0104] Electrophoresis results as follows Figure 3 As shown.

[0105] Fusion polymerases exhibit strong chain substitution capabilities at 10℃, 25℃, 30℃, 35℃, and 40℃, enabling them to perform rolling circle replication.

[0106] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A mutant PWT polymerase, characterized in that, The amino acid sequence of the mutant PWT polymerase is any one of A1) to A2): A1) The amino acid sequence shown in amino acids 407 to 986 of SEQ ID NO.1; A2) is an amino acid sequence obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in A1).

2. The mutant PWT polymerase according to claim 1, characterized in that, The label includes at least one of the following: labels that facilitate the dissolution, purification, and detection of mutant PWT polymerase.

3. The mutant PWT polymerase according to claim 1, characterized in that, The tags include maltose-binding protein and / or strep tags.

4. The mutant PWT polymerase according to claim 1, characterized in that, The amino acid sequence of the mutant PWT polymerase is shown in SEQ ID NO.

1.

5. Biomaterials related to the mutant PWT polymerase according to any one of claims 1 to 4, characterized in that: The biomaterial is any one of B1) to B4): B1) A nucleic acid molecule encoding the mutant PWT polymerase according to any one of claims 1 to 4; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

6. The biomaterial according to claim 5, characterized in that, The nucleotide sequence of the nucleic acid molecule is as shown in nucleotides 1219 to 2958 of SEQ ID NO.2 or SEQ ID NO.

2.

7. An enzyme preparation, characterized in that, Includes the mutant PWT polymerase as described in any one of claims 1 to 4.

8. The enzyme preparation according to claim 7, characterized in that, The enzyme preparation also includes at least one of nucleoside triphosphate and reaction buffer.

9. A method for preparing the mutant PWT polymerase according to any one of claims 1 to 4, characterized in that, include: The coding gene of the mutant PWT polymerase according to any one of claims 1 to 4 is introduced into a biological cell to express the coding gene, thereby obtaining the mutant PWT polymerase.

10. A method for replicating, amplifying, or sequencing template DNA, characterized in that, include: Mix the mutant PWT polymerase according to any one of claims 1 to 4 with template DNA and reaction reagents; The reaction reagents include reaction buffer, primers, and nucleoside triphosphates.

11. The method according to claim 10, characterized in that, The reaction reagents also include BSA and salt.

12. The application of any one of C1) to C3) in any one of D1) to D4). C1) The mutant PWT polymerase according to any one of claims 1 to 4; C2), the biomaterial as described in claim 5 or 6; C3), the enzyme preparation according to claim 7 or 8; D1) Nucleic acid amplification; D2) Preparation of nucleic acid amplification products; D3), ​​sequencing; D4) Prepare sequencing products.

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