CN113373127B - Taq DNA polymerase mutant and application thereof - Google Patents

Abstract

The invention discloses a Taq DNA polymerase mutant and application thereof. The invention provides a Taq DNA polymerase single-site mutant with improved thermostability, which is obtained by carrying out single-site mutation in G79E, G80A, D177E, A V, E189P, S357A, A E or G504K on an amino acid sequence shown in SEQ ID No. 1. On the basis of single-site mutation, the invention provides a Taq DNA polymerase multi-site mutant with further improved thermostability. qPCR thermal stability detection shows that the single-site or multi-site mutant of the Taq DNA polymerase provided by the invention has obviously improved enzyme activity compared with wild type Taq DNA polymerase, and the thermal stability is obviously superior to that of the wild type Taq DNA polymerase. The invention also provides application of the mutant as Taq DNA polymerase in gene amplification.

Description

Taq DNA polymerase mutant and application thereof

The application is a divisional application of a parent application with the invention name of Taq DNA polymerase mutant and application thereof, the application number is 202110342666.5, and the application date is 2021, 3 months and 30 days.

Technical Field

The invention relates to a polymerase mutant, in particular to a Taq DNA polymerase mutant with improved thermal stability and application thereof, belonging to the field of Taq DNA polymerase mutants.

Background

Taq DNA polymerase was the first discovered thermostable DNA polymerase, originally extracted from a strain of Thermobacter aquaticus (thermus aquaticus) isolated from hot springs by Saiki et al. The enzyme can resist high temperature, taq DNA polymerase can be used for DNA sequence determination in molecular cloning, and Polymerase Chain Reaction (PCR) can be used for in vitro amplification of specific fragments of DNA. During PCR, since Taq DNA polymerase is not inactivated during the denaturation step (about 94 ℃) and can directly enter the second cycle, it is not necessary to add new enzyme every cycle, which makes Taq DNA polymerase a unique enzyme in PCR reaction.

However, if the number of cycles is too large or the amplification temperature is high during the PCR amplification process, the enzyme activity of Taq DNA polymerase decreases, which leads to inaccurate amplification results, and thus, it is necessary to improve the thermal stability of Taq DNA polymerase.

Disclosure of Invention

One of the objects of the present invention would be to provide a Taq DNA polymerase single site mutant with improved thermostability;

the second object of the present invention is to provide Taq DNA polymerase multi-site mutants with improved thermostability;

the third object of the present invention is to provide a method for preparing the single-site mutant or the multi-site mutant of Taq DNA polymerase;

the fourth purpose of the invention is to apply the Taq DNA polymerase single site mutant or multi-site mutant to polymerization amplification.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

as a specific embodiment of the invention, the invention provides a Taq DNA polymerase single-site mutant with improved thermostability, which is obtained by carrying out single-site mutation on any one amino acid of G79E, G80A, D E, A V, E189P, S357A, A E or G504K on the amino acid sequence shown in SEQ ID No. 1; preferably, the mutant is obtained by carrying out single-site mutation on the amino acid sequence shown in SEQ ID No.1 by using any one of G79E, A V or G504K; more preferably, the amino acid sequence shown in SEQ ID No.1 is subjected to single-site mutation of any one of A180V or G504K.

The single-site mutant 'G79E' of the invention means that the 79 th amino acid of Taq DNA polymerase with the amino acid sequence shown as SEQ ID NO.1 is mutated into glutamic acid (E) from glycine (G); the expression of the remaining single-site mutations of the invention is analogized.

As a specific embodiment of the present invention, the present invention provides a Taq DNA polymerase multi-site mutant with improved thermostability, the multi-site mutant being selected from any one of the following (a) to (d):

(a) Mutant (A180V/G504K) obtained by simultaneously carrying out mutation on two sites of A180V and G504K on the amino acid sequence shown in EQ ID No. 1;

(b) A mutant (G79E/E189P) obtained by simultaneously carrying out mutation on two sites of G79E and E189P on an amino acid sequence shown in EQ ID No. 1;

(c) A mutant (A180V/G504K/G79E/E189P) obtained by simultaneously carrying out mutation on four sites of A180V, G32504K, G E and E189P on the amino acid sequence shown in EQ ID No. 1;

(d) The amino acid sequence shown in EQ ID No.1 is subjected to mutation at six sites of A180V, G504K, G E, E189P, D E and G80A at the same time to obtain a mutant (A180V/G504K/G79E/E189P/D177E/G80A).

The multi-site mutant 'A180V/G504K' of the invention refers to that alanine (A) at the 180 th site of Taq DNA polymerase with the amino acid sequence of SEQ ID NO.1 is changed into valine (V) and glycine (G) at the 504 th site is changed into lysine (K); the expression of the multi-site mutations of other said amino acids of the invention is analogized.

The encoding gene of the Taq DNA polymerase single-site mutant or multi-site mutant also belongs to the protection scope of the invention.

The invention also discloses a recombinant expression vector or a recombinant host cell containing the encoding gene of the Taq DNA polymerase single-site mutant or multi-site mutant; wherein, the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

The present invention further provides a method for preparing any one of the Taq DNA polymerase mutants, comprising:

(1) Operably connecting the encoding gene of the Taq DNA polymerase mutant with an expression regulation and control element to construct a recombinant expression vector;

(2) Transforming the recombinant expression vector into a host cell, culturing the host cell, inducing and expressing the recombinant protein, and purifying to obtain the recombinant protein.

The invention also discloses the application of the Taq DNA polymerase mutant as DNA polymerase in gene amplification.

Detailed description of the invention

Design, screening and enzymology performance detection of TaqDNA polymerase single-site mutant

In order to further improve the thermal stability of the Taq enzyme, 23 single-point mutations of the Taq enzyme are obtained according to methods such as gene consensus, AI model consensus design and the like, after the 23 single-point mutants are successfully constructed, the wild type and the mutants of the Taq enzyme are respectively subjected to heat treatment, wherein the heat treatment conditions are as follows: 30S at 95 ℃; 30S at 60 ℃; 60S at 72 ℃, and different numbers of heat treatment cycles are set. The control group was prepared without heat treatment. Then carrying out normal PCR reaction at 95 ℃ for 5min; 30S at 95 ℃, 30S at 60 ℃ and 60S at 72 ℃; 10min at 72 ℃. In order to compare the mutants more accurately by the gel electrophoresis of nucleic acids, the PCR reaction was set to 30 cycles in total. The previous experiment indicates that the previous 11 single-point mutants (H28Y, A61Y, G79E, A3597P, A109P, D E, A180P, T V, D237E, A472E, G K) soaking treatment 30 cycles are repeated for three times, and the result shows that the bands of the single-point mutation G79E, D34177E, A P, A472E and G504K are lighter than the wild type. However, since the number of heat treatment cycles is too large, the effect is not obvious, and the parallelism is poor, the five single-point mutations are re-tested. As shown in fig. 2, after 10 cycles of heat treatment, a180P activity was lost, thus discarding this single point mutant. Finally, G79E, D32177E, A E and G504K were selected for the design of subsequent multi-point mutations.

The last 12 single point mutants (D60K, G80A, A V, A180V, E P, L224 5657 zxft 56233K, H235 82 zxft 3282L, D244E, A293 3638 zxft 36357A) after heat treatment for different numbers of cycles respectively, after normal PCR reaction, the result shows that the single point mutant G80A, A180V, E49189P and S357A bands are brighter than the wild type, which indicates that the thermal stability is better than the wild type, so the four single point mutations are selected for subsequent multi-point mutation design.

Design, screening and enzymatic performance detection of TaqDNA polymerase multi-site mutant

Designing multi-point mutation according to the result of single-point mutation; after the heat stability test, the results show that the Taq enzyme multi-point mutants TD2 (A180V/G504K), TD3 (G79E/E189P), TD5 (A180V/G504K/G79E/E189P) and TD6 (A180V/G504K/G79E/E189P/D177E/G80A) have brighter bands than the wild type after heat treatment, which indicates that the heat stability is higher than that of the wild type. Therefore, the present invention selects these four multi-point mutants for qPCR thermostability testing. The results show that the enzyme activities of TD2, TD5 and TD6 are improved to different degrees compared with the wild type enzyme activities, wherein the enzyme activities of TD2 and TD6 are improved by more than two times, and in addition, after 10 cycles and 20 cycles of heat treatment, the residual enzyme activity of TD2 is obviously higher than that of the wild type enzyme. Although the enzyme activity of TD3 is slightly lower than that of the wild type, after 20 cycles of heat treatment, the residual enzyme activity of TD3 is higher than that of the wild type, which shows that the thermal stability of the TD3 is better than that of the wild type.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol.chem.260:2605-2608 (1985); and Cassol et al (1992); rossolini et al, mol cell. Probes 8.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, the description for a polypeptide applies equally to the description of a peptide and to the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

The terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill in the art.

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

The term "operably linked" refers to a functional linkage between two or more elements that may be operably linked and may or may not be contiguous.

The term "transformation" refers to the genetic transformation of a polynucleotide or polypeptide into a host cell in such a manner that the encoding gene is introduced into the interior of the host cell.

The term "expression": transcription and/or translation of endogenous genes or transgenes in host cells.

The term "coding gene": a nucleic acid sequence transcribed into RNA.

Drawings

FIG. 1 is a diagram showing the results of nucleic acid electrophoresis in the detection of thermal stability of a single-point mutant of Taq enzyme (No. 1-11);

FIG. 2 is a diagram showing the result of nucleic acid electrophoresis in the detection of the thermal stability of single-point mutants of Taq enzyme;

FIG. 3 is a diagram showing the results of nucleic acid electrophoresis in the detection of thermal stability of single-point mutants of Taq enzyme (Nos. 12-23);

FIG. 4 is a diagram of the result of nucleic acid electrophoresis in the detection of the thermal stability of the Taq enzyme multi-point mutant.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the embodiments are illustrative only and are not to be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

Test example 1 design, screening and enzymatic Performance test of TaqDNA polymerase Single-site mutants

In order to further improve the thermostability of Taq enzyme, 23 single-point mutations of Taq enzyme were obtained by methods such as gene consensus and AI model consensus, as shown in table 1 below.

TABLE 1 Taq enzyme Single Point mutation site

After the 23 single-point mutants are successfully constructed, respectively carrying out heat treatment on the Taq enzyme wild type and the mutant, wherein the heat treatment conditions are as follows: 30S at 95 ℃; 30S at 60 ℃; 60S at 72 ℃, and different numbers of heat treatment cycles are set. The control group was prepared without heat treatment. Then carrying out normal PCR reaction at 95 ℃ for 5min; 30S at 95 ℃, 30S at 60 ℃ and 60S at 72 ℃; 10min at 72 ℃. In order to compare the mutants more accurately by the gel electrophoresis of nucleic acids, the PCR reaction was set to 30 cycles in total.

The previous experiments found that the first 11 single point mutants (H28Y, A61Y, G79E, A3597P, A109P, D E, A180P, T V, D237E, A472E, G K) soaked for 30 cycles, repeated three times, and showed that the bands of the single point mutation G79E, D E, A P, A472E and G504K were slightly brighter than the wild type (FIG. 1). However, since the number of heat treatment cycles was too large, the effect was not significant, and the parallelism was poor, these five single point mutations were re-tested. As shown in fig. 2, after 10 cycles of heat treatment, a180P activity was lost, thus discarding this single point mutant. Finally, G79E, D177E, A E and G504K were selected for subsequent multi-point mutation design.

The last 12 single point mutants (D60K, G80A, A V, A180V, E P, L224 5657 zxft 56233K, H235 82 zxft 3282L, D244E, A293 3638 zxft 36357A) after heat treatment for different numbers of cycles respectively, after normal PCR reaction, the results showed that the bands of the single point mutant G80A, A180V, E P and S357A are brighter than the wild type (FIG. 3), indicating that the thermal stability is better than the wild type, so the four single point mutations were selected for subsequent multi-point mutation design.

Test example 2 design, screening and enzymatic Performance testing of TaqDNA polymerase Multi-site mutants

Based on the single point mutation results of test example 1, a multi-point mutation was designed, and the designed multi-point mutation sites are shown in Table 2.

TABLE 2 Taq enzyme multiple site mutations

The results of the thermal stability test showed that the Taq enzyme multipoint mutants TD2, TD3, TD5 and TD6 were brighter than the wild type after heat treatment (FIG. 4), indicating that the thermal stability was higher than that of the wild type.

Therefore, the present assay selects these four multi-point mutants for qPCR thermostability testing. The thermal stability detection results are shown in table 3, TD2, TD5 and TD6 have different degrees of improvement compared with wild type enzyme activity, wherein the enzyme activity of TD2 and TD6 is improved by more than two times, and in addition, after 10 cycles and 20 cycles of heat treatment, the residual enzyme activity of TD2 is significantly higher than that of wild type. Although the enzyme activity of TD3 is slightly lower than that of the wild type, the residual enzyme activity of TD3 is higher than that of the wild type after 20 cycles of heat treatment, which shows that the thermal stability of the wild type is better than that of the wild type.

TABLE 3 Taq enzyme multiple point mutant thermostability assay qPCR results

SEQUENCE LISTING

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Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly

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Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala

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Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly

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Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val

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Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro

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Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu

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Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly

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Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala

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Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His

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Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala

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Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro

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Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu

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Claims (7)

1. A Taq DNA polymerase mutant with improved thermostability, which is characterized in that the mutant is obtained by carrying out G79E amino acid single site mutation on an amino acid sequence shown as SEQ ID No. 1.

2. A Taq DNA polymerase mutant with improved thermostability, which is characterized in that the mutant is obtained by simultaneously carrying out mutation on two sites of G79E and E189P on an amino acid sequence shown in EQ ID No. 1.

3. A gene encoding the Taq DNA polymerase mutant according to claim 1 or 2.

4. A recombinant expression vector comprising the gene of claim 3.

5. A recombinant host cell comprising the recombinant expression vector of claim 4.

6. A method for preparing the Taq DNA polymerase mutant according to claim 1 or 2, comprising the steps of:

(1) Operably connecting the encoding gene of the Taq DNA polymerase mutant with an expression regulation and control element to construct a recombinant expression vector;

(2) Transforming the recombinant expression vector into a host cell, culturing the host cell, inducing and expressing the recombinant protein, and purifying to obtain the recombinant protein.

7. The use of the Taq DNA polymerase mutant of claim 1 or 2 as Taq DNA polymerase in gene amplification.

Images