CN112725301A

CN112725301A - Taq DNA polymerase mutant and application thereof

Info

Publication number: CN112725301A
Application number: CN202110342666.5A
Authority: CN
Inventors: 关菲菲; 田�健; 伍宁丰; 杨丽鑫; 刘晓青
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2021-04-30
Anticipated expiration: 2041-03-30
Also published as: CN113373127B; CN112725301B; CN113373127A

Abstract

The invention discloses a Taq DNA polymerase mutant and its application. The present invention provides a single-site mutant of Taq DNA polymerase with improved thermostability, which is obtained by subjecting the amino acid sequence shown in SEQ ID No. 1 to G79E, G80A, D177E, A180V, E189P, S357A, A472E or G504K mutants obtained by single-site mutation in . On the basis of single-site mutation, the present invention provides a Taq DNA polymerase multi-site mutant with further improved thermal stability. The qPCR thermal stability test shows that the single-site or multi-site mutants of the Taq DNA polymerase provided by the present invention have significantly improved enzyme activity compared with the wild-type enzyme, and the thermal stability is obviously better than that of the wild-type Taq DNA polymerase. The present invention also provides the use of the mutant as Taq DNA polymerase in gene amplification.

Description

Taq DNA polymerase mutant and application thereof

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 found 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 of Taq DNA polymerase is too large or the amplification temperature is high during PCR amplification, the enzyme activity is reduced, which leads to inaccurate amplification results, and thus, it is necessary to improve the thermostability 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 thermal stability;

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

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

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 present invention, the present invention provides a Taq DNA polymerase single-site mutant with improved thermostability, which is a mutant obtained by subjecting the amino acid sequence shown in SEQ ID No.1 to any one of the amino acid single-site mutations of G79E, G80A, D177E, A180V, E189P, S357A, A472E or G504K; preferably, the mutant is obtained by subjecting the amino acid sequence shown in SEQ ID No.1 to single-site mutation of any one of the amino acid sequences G79E, A180V 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 to obtain a mutant.

The single-site mutant 'G79E' of the invention refers to that the 79 th amino acid of Taq DNA polymerase with the amino acid sequence shown in SEQ ID NO.1 is mutated from glycine (G) to glutamic acid (E); 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) 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 the amino acid sequence shown in EQ ID No.1 at two sites of G79E and E189P;

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

(d) a mutant obtained by carrying out mutation on six sites of A180V, G504K, G79E, E189P, D177E and G80A on the amino acid sequence shown in EQ ID No.1 at the same time (A180V/G504K/G79E/E189P/D177E/G80A).

The multi-site mutant "A180V/G504K" of the present invention means that alanine (A) at position 180 of Taq DNA polymerase having the amino acid sequence shown in SEQ ID NO.1 is mutated to valine (V) and glycine (G) at position 504 is mutated to 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) connecting the encoding gene of the Taq DNA polymerase mutant with an expression regulation element in an operable manner 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 by methods such as gene consensus and AI model co-design, after the 23 single-point mutants are successfully constructed, the wild type and the mutant of the Taq enzyme are respectively subjected to heat treatment under the following conditions: 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 5 min; 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. Previous experiments have found that the first 11 single-point mutants (H28Y, A61Y, G79E, A97P, A109P, D177E, A180P, T186V, D237E, A472E and G504K) are soaked for 30 cycles and repeated for three times, and the results show that the single-point mutations G79E, D177E, A180P, A472E and G504K have lighter bands 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, a180P activity was lost after 10 cycles of heat treatment, thus discarding this single point mutant. Finally, G79E, D177E, a472E and G504K were selected for the design of subsequent multiple mutations.

After the last 12 single-point mutants (D60K, G80A, A155V, A180V, E189P, L224V, L233K, H235F, M236L, D244E, A293P and S357A) are respectively subjected to heat treatment for different numbers of cycles, and normal PCR reaction is carried out, the result shows that the single-point mutants G80A, A180V, E189P and S357A have brighter bands than the wild type, which indicates that the thermal stability is better than that of the wild type, so that the four single-point mutations are selected for designing the subsequent multiple-point mutation.

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 thermal stability test is carried out on 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) are subjected to heat treatment, the bands are brighter than the wild type, and the thermal 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 wild type enzyme activities, wherein the enzyme activities of TD2 and TD6 are improved by more than two times, and in addition, after 10-cycle and 20-cycle 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 indicates that the thermal stability 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. In particular, 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-,Mol Cell. Probes 8:91-98 (1994)）。

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 an endogenous gene or transgene in a host cell.

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 described embodiments are exemplary only and are not limiting upon the scope of the invention. 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 modifications may 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 in total by methods such as gene consensus and AI model co-design, as shown in table 1 below.

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 5 min; 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.

Previous experiments revealed that the first 11 single-point mutants (H28Y, a61Y, G79E, a97P, a109P, D177E, a180P, T186V, D237E, a472E, G504K) were soaked for 30 cycles, which were repeated three times, and the results showed that the bands of the single-point mutations G79E, D177E, a180P, 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, a180P activity was lost after 10 cycles of heat treatment, thus discarding this single point mutant. Finally, G79E, D177E, a472E and G504K were selected for the design of subsequent multiple mutations.

After the last 12 single-point mutants (D60K, G80A, a155V, a180V, E189P, L224V, L233K, H235F, M236L, D244E, a293P and S357A) were subjected to heat treatment for different numbers of cycles respectively and subjected to normal PCR reaction, the results showed that the bands of the single-point mutants G80A, a180V, E189P and S357A were brighter than the wild type (fig. 3), indicating that the thermostability was better than the wild type, and therefore, the four single-point mutations were selected for subsequent multi-point mutation design.

Experimental 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.

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 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, the enzyme activities of TD2, TD5 and TD6 are improved to different degrees compared with the wild type enzyme, 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 significantly 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 indicates that the thermal stability is better than that of the wild type.

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

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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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Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe

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Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp

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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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Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly

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Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr

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Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro

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

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Claims

1. a Taq DNA polymerase mutant with improved thermostability, characterized in that the mutant is the amino acid sequence shown in SEQ ID No.1 subjected to G79E, G80A, D177E, A180V, E189P, S357A, A472E or A mutant obtained by site mutation of any amino acid in G504K.

2. The Taq DNA polymerase mutant according to claim 1, wherein the mutant is a single point mutation of any amino acid in G79E, A180V or G504K for the amino acid sequence shown in SEQ ID No. 1 obtained mutants.

3. The Taq DNA polymerase mutant according to claim 2, wherein the mutant is obtained by performing a single point mutation of any amino acid in A180V or G504K with the amino acid sequence shown in SEQ ID No. 1 mutants.

4. A Taq DNA polymerase mutant with improved thermostability, wherein the mutant is selected from any one of the following (a)-(d):

(a) A mutant obtained by simultaneously mutating the amino acid sequence shown in EQ ID No.1 at two sites, A180V and G504K;

(b) a mutant obtained by simultaneously mutating the amino acid sequence shown in EQ ID No.1 at two sites, G79E and E189P;

(c) a mutant obtained by simultaneously mutating the amino acid sequence shown in EQ ID No.1 at four sites, A180V, G504K, G79E and E189P;

(d) A mutant obtained by simultaneously mutating the amino acid sequence shown in EQ ID No. 1 at six sites A180V , G504K , G79E , E189P, D177E and G80A.

5. The Taq DNA polymerase mutant according to claim 4, wherein the mutant is selected from any one of (a) or (d).

6. A gene encoding the Taq DNA polymerase mutant of any one of claims 1 to 5.

7. A recombinant expression vector containing the gene of claim 6.

8. A recombinant host cell comprising the recombinant expression vector of claim 7.

9. a method for preparing the Taq DNA polymerase mutant described in any one of claims 1 to 5, is characterized in that, comprises the following steps:

(1) operably linking the encoding gene of the Taq DNA polymerase mutant with an expression control element to construct a recombinant expression vector;

(2) Transforming the recombinant expression vector into host cells, culturing the host cells, inducing expression of the recombinant protein, and purifying, that is, it is obtained.

10. Use of the Taq DNA polymerase mutant of any one of claims 1 to 5 as Taq DNA polymerase in gene amplification.