CN116716274A

CN116716274A - Mutant Taq DNA polymerase and application thereof

Info

Publication number: CN116716274A
Application number: CN202310763569.2A
Authority: CN
Inventors: 齐欣; 宋新文; 耿亮; 辛文
Original assignee: Beijing Quanshijin Biotechnology Co ltd
Current assignee: Beijing Quanshijin Biotechnology Co ltd
Priority date: 2023-06-27
Filing date: 2023-06-27
Publication date: 2023-09-08

Abstract

The invention discloses mutant Taq DNA polymerase and application thereof. The invention firstly discloses a mutant Taq DNA polymerase, which has the following amino acid substitution at one amino acid position in the sequence shown in SEQ ID NO.1, wherein the substitution is expressed by a triplet: letter-number-letter, wherein the number indicates the position of the mutated amino acid, the letter preceding the number corresponds to the amino acid to which the mutation relates, and the letter following the number indicates the amino acid used to replace the amino acid preceding the number: a705E or a705K. The invention further discloses application of the polymerase in the PCR field of samples containing humic acid and/or humic acid analogues. The polymerase provided by the invention can tolerate humic acid with higher concentration due to conformational change, greatly improves the amplification capability of the soil microorganism extract, and has great value in the fields of detection of soil microorganism diversity, monitoring of environmental pathogenic microorganisms and the like.

Description

Mutant Taq DNA polymerase and application thereof

Technical Field

The present invention relates to the field of biotechnology. More particularly, relates to a mutant Taq DNA polymerase and application thereof.

Background

Soil is a main place where microorganisms inhabit, and the distribution and life activities of microorganisms in the soil are closely related to the conditions of soil fertility, plant nutrition, plant diseases and the like, so that research and classification identification of soil microorganisms by utilizing the morphology, culture characteristics, physiological and biochemical characteristics and genetic characteristics of microorganisms are an important method for researching soil environment. However, the soil microorganisms are difficult to separate and identify due to the limitation of 85% -99% of various conditions, so that the soil microorganism research is limited to a certain extent. The separation and purification detection method of microbiology by using the traditional method can greatly underestimate the diversity of microorganisms in a sample and ignore some microorganism types with extremely low content. Therefore, the application of the molecular biological method based on the PCR technology and the research of soil organisms make up the defects of the traditional microbiological research.

The physical and chemical properties of the soil are extremely complex, and the crude extracted soil DNA contains inhibitory pollutants such as humic acid, phenolic compounds, heavy metal ions and the like. Among these, the inhibition of humic acid and humic acid analogues is most pronounced, and they interfere with normal PCR amplification systems by interfering with cleavage, degrading nucleic acids or non-specific adsorption and inhibiting enzymatic activity. Humic acid is tightly bound to DNA during extraction of soil DNA, it is difficult to separate, and a very low concentration of humic acid (0.08. Mu.g/ml) is sufficient to inhibit the activity of DNA polymerase. Therefore, the inhibition effect of humic acid is reduced, and the method becomes a key for researching the diversity of soil microorganisms. At present, many researches on the bacterial species of activated sludge in soil and water environment are carried out, and accurate qualitative and quantitative determination is required by a fluorescent quantitative PCR technical method. The premise of the accuracy of the fluorescent quantitative PCR is that microorganisms in the activated sludge are extracted as completely as possible, the template is good in quality and high in purity, and the inhibitor existing in the activated sludge is removed as cleanly as possible.

In order to realize the amplification of sample DNA in soil, technical means are generally required to remove humic acid residues in the sample, the humic acid removal process is complicated, and the effective methods for removing humic acid at present mainly comprise membrane technology, ion exchange, catalytic oxidation and the like, and the methods have the defects that other steps are required to be introduced and other pollution may be introduced in the removal process. Therefore, when the soil direct expansion product is developed, the concentration of humic acid can be reduced by a dilution method, and meanwhile, the concentration of DNA can be diluted to influence the experimental result. Other extraction methods for removing humic acid are very complex and the effect cannot be guaranteed. In order to simplify the experimental process, the enzyme itself can endure the inhibition effect of humic acid with higher concentration through the transformation of DNA polymerase, thereby reducing the preparation requirement of DNA sample, and the transformed enzyme can be applied to the metagenome direct expansion experiment, thereby simplifying the experimental steps and improving the experimental efficiency.

Taq DNA polymerase is found in a Thermus aquaticus (Thermus aquaticus) isolated from volcanic spa, and thus has extremely high thermal stability, can withstand the thermal denaturation step in the PCR process, and becomes the first generation of thermostable DNA polymerase. Due to the characteristics of stability, easy expression and purification, strong amplification efficiency and the like, taq DNA polymerase is widely applied to fluorescent quantitative PCR. However, wild Taq DNA polymerase has weaker tolerance to humic acid, and the polymerase activity is obviously inhibited in the presence of humic acid with lower concentration, and finally the experimental result is influenced.

Therefore, in order to realize the amplification of DNA samples in complex environments, a series of modifications are required to be made to wild-type Taq DNA polymerase, so that the performances of the wild-type Taq DNA polymerase in terms of environmental tolerance, stability, catalytic activity and the like are further improved.

Disclosure of Invention

The invention aims to provide a mutant of Taq DNA polymerase, which has obviously improved tolerance to humic acid and humic acid analogues compared with wild Taq DNA polymerase.

It is another object of the present invention to provide the use of mutants of Taq DNA polymerase as described above in the field of PCR of samples comprising humic acid and/or humic acid analogues.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention firstly provides a mutant Taq DNA polymerase, which has the following amino acid substitution at one amino acid position in the sequence shown in SEQ ID NO.1, wherein the substitution is expressed by a triplet: letter-number-letter, wherein the number indicates the position of the mutated amino acid, the letter preceding the number corresponds to the amino acid to which the mutation relates, and the letter following the number indicates the amino acid used to replace the amino acid preceding the number: a705E or a705K.

Further, the mutant Taq DNA polymerase is a protein as shown in any one of the following:

a1 A protein consisting of the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO. 5;

a2 A protein derived from a 1) having the mutant Taq DNA polymerase activity by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO. 5;

a3 A protein having the mutant Taq DNA polymerase activity, which has 80% identity or more with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO. 5.

Preferably, the 80% and above identity is 85% identity, more preferably 90% identity, most preferably 95% identity.

The nucleotide sequence encoding the mutant Taq DNA polymerase is also within the scope of the present invention.

Further, the nucleotide sequence of the coding mutant Taq DNA polymerase is shown as SEQ ID NO.4 or SEQ ID NO. 6.

Recombinant vectors or recombinant cells comprising the above-described nucleotide sequences are also within the scope of the present invention.

In a specific embodiment of the invention, the recombinant vector is recombinant vectors pET-21a-TaqA705E and pET-21a-TaqA705K; wherein, the recombinant vector pET-21a-TaqA705E is obtained by connecting a nucleotide sequence shown in SEQ ID NO.4 of encoding mutant Taq DNA polymerase A705E between EcoR I and Sal I cleavage sites of pET-21a, and keeping other sequences of pET-21a unchanged; the pET-21a-TaqA705K is obtained by connecting a nucleotide sequence shown in SEQ ID NO.6 of encoding mutant Taq DNA polymerase A705K between EcoR I and Sal I cleavage sites of pET-21a, and keeping other sequences of pET-21a unchanged.

Further, the host cell of the recombinant cell is an engineered BL21 E.coli. In a specific embodiment of the invention, the host cell is a BL21 (DE 3) competent cell.

The invention further discloses application of the mutant Taq DNA polymerase, the nucleotide sequence or the recombinant vector or the recombinant cell in any one of the following:

b1 Application in the field of biotechnology;

b2 Use in the field of PCR;

b3 In the field of PCR of samples comprising humic acid and/or humic acid analogues.

Further, the final concentration of humic acid in the sample is not more than 25 ng/. Mu.l; preferably, it is not more than 20 ng/. Mu.l.

The original amino acid sequence of the mutant Taq DNA polymerase is shown as SEQ ID NO.1, and the gene sequence of the mutant Taq DNA polymerase comprises a nucleotide sequence shown as SEQ ID NO.2, wherein the amino acid shown as SEQ ID NO.1 is derived from Taq DNA polymerase (Thermus aquaticus). Alanine at position 705 was randomly mutated to any amino acid by the design of the degenerate primer. Through protein expression and screening, two mutant proteins are screened, and can both tolerate high-concentration humic acid in a PCR system, namely A705E and A705K, the amino acid sequences of the two mutant proteins are respectively shown as SEQ ID NO.3 and SEQ ID NO.5, and the gene sequences of the two mutant proteins are respectively shown as SEQ ID NO.4 and SEQ ID NO. 6.

The beneficial effects of the invention are as follows:

the mutant Taq DNA polymerase changes the conformation of the polymerase by carrying out amino acid substitution on the A705 locus on the wild Taq DNA polymerase, improves the indexes such as amplification efficiency, amplification length and the like under the same conditions, improves the tolerance of the Taq DNA polymerase to humic acid and humic acid analogues, can tolerate humic acid with higher concentration, improves the tolerance of the humic acid from 5 ng/mu l to more than 25 ng/mu l, still maintains higher enzyme activity, greatly improves the amplification capability of soil microorganism extracts, and has great value in the fields of detection of soil microorganism diversity, monitoring of environmental pathogenic microorganisms and the like. In addition, the mutant Taq DNA polymerase can realize DNA amplification experiments in complex samples, and can also be directly applied to DNA direct amplification experiments.

Drawings

The following describes the embodiments of the present invention in further detail with reference to the drawings.

FIG. 1 is a graph showing the results of pressure screening of wild-type Taq DNA polymerase (WT) with mutant Taq enzyme under the stress of humic acid, wherein: lane M:plus II DNA Marker (Transgen Biotech); lane 1: PCR products of WT in normal system; lane 2: PCR products of WT containing 10ng/ul humic acid in the system; lane 3: PCR products of WT containing 30ng/ul humic acid in the system; lane 4: PCR products of WT containing 40ng/ul humic acid in the system; lane 5: PCR products of WT containing 50ng/ul humic acid in the system; lane 6: PCR products of mutant Taq enzyme in a normal system; lane 7: PCR products of mutant Taq enzyme containing 10ng/ul humic acid in the system; lane 8: PCR products of mutant Taq enzyme containing 30ng/ul humic acid in the system; lane 9: PCR products of mutant Taq enzyme containing 40ng/ul humic acid in the system; lane 10: PCR products of mutant Taq enzyme containing 50ng/ul humic acid in the system.

FIG. 2 is a PAGE gel of wild-type Taq DNA polymerase (WT) purified with mutant Taq DNA polymerases A705E and A705K.

FIG. 3 is an agarose gel diagram of the amplification of 850kp long genes using Hela cell genomic DNA as a template in the presence of humic acid at different concentrations by purified wild-type Taq DNA polymerase (WT) and mutant Taq DNA polymerases A705E and A705K.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

The primers used in the following examples are shown in Table 1:

TABLE 1 primer sequences

Remarks: n= A, T, C or G.

EXAMPLE 1 obtaining of humic acid-tolerant mutant Taq DNA polymerase

The NCBI and the protein database PDB are searched to obtain the protein sequence (shown as SEQ ID NO.1, the gene sequence comprises the nucleotide sequence shown as SEQ ID NO. 2) and the structure of mutant Taq DNA polymerase, alanine (Ala, A) at 705 site is selected for saturation mutation transformation through analysis and comparison of the sequence and the structure to form a library containing a plurality of mutant plasmids, the library is screened by adding screening pressure, and finally the mutant Taq DNA polymerase with humic acid tolerance is screened.

The method comprises the following specific steps:

1. construction of mutant libraries

Primers were designed according to the sequence shown in SEQ ID No.2, and a restriction enzyme site EcoR I/Sal I was added to the primers. The method comprises the following specific steps: and (3) performing PCR amplification by using the sequence shown in SEQ ID NO.2 as a template and using primers Taq-1 and Taq-832 to obtain a PCR product. The PCR product was gel-recovered, digested with EcoR I/Sal I (from Transgen Biotech), ligated with linearization vector (pET-21 a, ecoR I/Sal I double digested), and transferred to Trans1-T1 competence (from Transgen Biotech), plated on ampicillin-resistant plates, and incubated overnight at 37 ℃. The monoclonal colony on the plate is picked up the next day, plasmid extraction is carried out, and the plasmid is determined to be pET-21a-Taq cloning plasmid after the sequencing is correct.

The pET-21a-Taq (comprising the gene sequence of Taq DNA polymerase shown in SEQ ID NO. 2) cloning plasmid was used as a template, usingFastPfu PCRSuperMix (Transgen Biotech) enzyme PCR amplification of the mutant library was performed to obtain PCR products. Wherein, the PCR amplification system is shown in Table 2, and the amplification cycle steps are as follows: 94℃for 5min,94℃for 30s, 55℃for 30s, 72℃for 2min, 30 cycles total, 72℃for 10min, and the process is completed.

TABLE 2 PCR amplification System of mutant library (50. Mu.l System)

And (5) carrying out agarose gel identification and gel recovery on the PCR product. Mu.l of PCR product was added to 50. Mu.l of clone competent cells, plated on ampicillin-resistant plates and incubated overnight at 37 ℃. The colonies on the plates were collected the next morning and plasmid extraction was performed to obtain the constructed mutant library plasmids.

2. Expression of mutant Taq enzyme

The mutant library plasmids were transformed into Trans 5. Alpha. Chemically competent cells (purchased from Transgen Biotech), incubated on ice for half an hour followed by heat shock at 42℃for 30s, incubated for 1 hour with SOC, plated, and incubated overnight at 37 ℃. The next day, the monoclonal cells were collected into 10ml of LB (peptone 10g/L, yeast powder 5g/L, naCl 10 g/L), and cultured at 37℃at 220rpm until OD600 was 0.6-0.8, and then induced by adding IPTG for 5 hours, and then collected at 3000 rpm.

The thalli are suspended by 500 mul of 10 XTaq PCR buffer solution, and supernatant liquid is collected after ultrasonic crushing, thus obtaining the crude extract of mutant Taq enzyme.

3. Screening of mutant Taq enzymes

And (3) carrying out gradient dilution on humic acid to 100 ng/. Mu.l to 500 ng/. Mu.l, adding the humic acid into a PCR amplification system as required to serve as screening pressure for PCR amplification, obtaining a PCR product, and screening mutant Taq enzyme. And taking a PCR product obtained by wild Taq DNA polymerase in a corresponding PCR amplification system as a control.

The template required for detection is pET-21a-Taq (comprising the gene sequence of Taq DNA polymerase shown in SEQ ID NO. 2) cloning plasmid, the PCR amplification system is shown in Table 3, and the amplification cycle steps are as follows: 94℃for 5min,94℃for 30s, 55℃for 30s, 72℃for 2min, 30 cycles total, 72℃for 10min, and the process is completed.

TABLE 3 PCR amplification System for humic acid screening (25. Mu.l System)

The PCR products were subjected to agarose gel identification, and the results are shown in FIG. 1, and the results show that the tolerance of mutant Taq DNA polymerase to humic acid is significantly higher than that of wild-type (WT) Taq DNA polymerase. Clones of the gel recovery target bands were sequenced, and the sequencing results showed that, by pressure screening of humic acid, alanine (WT, wild type) was 19 times in position 705, glutamic acid (a 705E) was 37 times, lysine was 27 times (a 705K)) and aspartic acid was 13 times, indicating that a705E and a705K could be subjected to further validation experiments. In contrast, alanine, glutamic acid, lysine, aspartic acid, histidine, glutamine, cysteine, threonine, leucine, isoleucine, glycine, etc. were each present 2-3 times without humic acid pressure screening, and the number of repetitions was much smaller than the number of humic acid pressure screening.

3. Tolerance test of mutant enzymes to humic acid

1. Expression and purification of mutant Taq DNA polymerase A705E and A705K

The genes encoding Taq DNA polymerase mutants A705E and A705K were each double digested (EcoR I/Sal I) and ligated with linearization vector (pET-21 a, ecoR I/Sal I double digested) and transformed into Trans1-T1 competence (purchased from TransGen Biotech), spread on ampicillin-resistant plates and incubated overnight at 37 ℃. The next day, the monoclonal colonies on the plates were picked, plasmid extracted, and the mutant clone plasmids pET-21a-TaqA705E and pET-21a-TaqA705K were determined after the sequencing was correct. After transformation of the mutant cloning plasmid into BL21 (DE 3) competent cells (purchased from TransGen Biotech), they were plated onto ampicillin-resistant plates and incubated overnight at 37 ℃. The following day, monoclonal colonies on the plates were picked and grown up. Single colonies were inoculated into 10ml of LB medium, cultured at 37℃at 220rpm until the OD600 was about 1, inoculated into 1L of LB medium, cultured at 37℃at 220rpm until the OD600 was about 0.6, and added with IPTG at a final concentration of 0.5 mM. Inducing at 37 deg.c for 5 hr and centrifuging to collect thallus. And (3) after the thalli are crushed, centrifuging and collecting supernatant, namely, a crude extract of the Taq DNA polymerase mutant, carrying out affinity chromatography on the crude extract, collecting a purified product, and carrying out polyacrylamide gel detection, wherein the results are shown in figure 2, and wild Taq DNA polymerase, mutant Taq DNA polymerase A705E and A705K are respectively obtained.

2. Detection of the tolerance of mutant enzymes to humic acid

Humic acid with different concentrations is added into human genome DNA (Hela cell genome DNA), and PCR amplification is carried out by using wild Taq DNA polymerase, mutant Taq DNA polymerase A705E and A705K respectively and using the Hela cell genome as a template, so as to obtain PCR products. Wherein, the PCR amplification system is shown in Table 4, and the amplification cycle steps are as follows: 94℃for 5min,94℃for 30s, 55℃for 30s, 72℃for 2min, 30 cycles total, 72℃for 10min, and the process is completed. And (3) performing PCR amplification by using the wild Taq DNA polymerase under the condition of no humic acid and using the Hela cell genome DNA as a template to obtain a PCR product serving as a positive control.

Agarose gel identification is carried out on the PCR product, and the result is shown in figure 3, and the mutant Taq DNA polymerase A705E has the best anti-humic acid inhibition effect; mutant Taq DNA polymerase A705K times, wild-type Taq DNA polymerase was worst.

TABLE 4 PCR amplification System for tolerance test of Taq DNA polymerase to humic acid

Amplification tests of other inhibitors such as blood, EDTA, protease, etc., were performed on these two mutant Taq DNA polymerases A705E and A705K, and no enhancement in tolerance was found, indicating specific anti-humic acid inhibition by mutant Taq DNA polymerases A705E and A705K.

In conclusion, mutant Taq DNA polymerases A705E and A705K capable of tolerating humic acid are finally obtained, wherein the amino acid sequence of A705E is shown as SEQ ID NO.3, and the gene sequence is shown as SEQ ID NO. 4; the amino acid sequence of A705K is shown as SEQ ID NO.5, and the gene sequence is shown as SEQ ID NO.6, and can be used in the PCR field of samples containing humic acid or analogues thereof.

It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A mutant Taq DNA polymerase, characterized in that the mutant Taq DNA polymerase has an amino acid substitution at one of the following amino acid positions in the sequence shown in SEQ ID No.1, said substitution being represented by a triplet: letter-number-letter, wherein the number indicates the position of the mutated amino acid, the letter preceding the number corresponds to the amino acid to which the mutation relates, and the letter following the number indicates the amino acid used to replace the amino acid preceding the number: a705E or a705K.

2. The mutant Taq DNA polymerase of claim 1, wherein the mutant Taq DNA polymerase is a protein as set forth in any one of:

3. A nucleotide sequence encoding the mutant Taq DNA polymerase of claim 1 or 2.

4. A nucleotide sequence according to claim 3, wherein the mutant Taq DNA polymerase has the nucleotide sequence shown in SEQ ID No.4 or SEQ ID No. 6.

5. A recombinant vector comprising the nucleotide sequence of claim 3 or 4.

6. A recombinant cell comprising the nucleotide sequence of claim 3 or 4.

7. Use of a mutant Taq DNA polymerase according to claim 1 or 2, a nucleotide sequence according to claim 3 or 4, a recombinant vector according to claim 5, or a recombinant cell according to claim 6 in the field of biotechnology.

8. Use of a mutant Taq DNA polymerase according to claim 1 or 2, a nucleotide sequence according to claim 3 or 4, a recombinant vector according to claim 5, or a recombinant cell according to claim 6 in the field of PCR.

9. Use of a mutant Taq DNA polymerase according to claim 1 or 2, a nucleotide sequence according to claim 3 or 4, a recombinant vector according to claim 5, or a recombinant cell according to claim 6 in the field of PCR of samples comprising humic acid and/or humic acid analogues.

10. The use according to claim 9, wherein the final concentration of humic acid in the sample is not more than 25 ng/. Mu.l; preferably, it is not more than 20 ng/. Mu.l.