CN117660394A

CN117660394A - DNA polymerase mutant with high amplification activity

Info

Publication number: CN117660394A
Application number: CN202311686872.3A
Authority: CN
Inventors: 蒋析文; 刘霭珊
Original assignee: Guangzhou Da'an Gene Co ltd
Current assignee: Guangzhou Da'an Gene Co ltd
Priority date: 2019-01-29
Filing date: 2019-01-29
Publication date: 2024-03-08
Also published as: CN111484987A; CN117660396A; CN111484987B; CN117660400A; CN117660401A; CN117660407A; CN117778347A; CN117660404A; CN117757767A; CN117660402A; CN117660410A; CN117660406A; CN117660399A; CN117660408A; CN117660398A; CN117660405A; CN117660395A; CN117660397A; CN117660409A; CN117660403A

Abstract

The invention provides a DNA polymerase mutant with high amplification activity, in particular to a method for constructing a random mutation library for a polymerase activity structural domain of Taq enzyme by applying a protein directed evolution technology, naturally eliminating unsuitable mutation by gradually adding screening pressure, gradually accumulating mutation with dominant character, finally screening a series of amino acid sites and mutation thereof which have key effects on the amplification performance and the polymerization performance of the Taq enzyme, and obtaining the Taq enzyme mutant with high amplification performance.

Description

DNA polymerase mutant with high amplification activity

The application is a divisional application of the invention patent application with the application date of 2019, 01 and 29, the application number of 201910083410.X and the invention creation name of 'a heat-resistant DNA polymerase mutant with high amplification activity'.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a heat-resistant DNA polymerase mutant with high amplification activity.

Background

Taq enzyme is a thermostable DNA polymerase derived from thermostable bacteria Thermus aquaticus, has a molecular weight of 94kDa, and has an optimum reaction temperature of 75-80℃and an active half-life of 40 minutes at 95℃in the presence of magnesium ions, and has 5'-3' exonuclease activity. Because of its high temperature resistance, it is widely used in Polymerase Chain Reaction (PCR), and is the first enzyme for nucleic acid amplification, detection and other reactions. Commercial Taq enzyme was cloned and expressed using E.coli prokaryotic expression system. Modern molecular biological detection technology has higher and higher requirements on sensitivity, accuracy and durability of PCR reaction, and wild type Taq enzyme can not completely meet the requirements of practical application. Many attempts have been made to mutate the Taq enzyme sequence to make it more amenable to the use of specific techniques, such as the addition of DNA binding domains to have greater elongation activity (Wang Y (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in v nucleic Acids Res 32, 1197-1207)); the domains were made more fidelity by site-directed mutagenesis, deletion (Suzuki M, yoshida S, adman ET, blank A, loeb LA (2000) Thermus Aquaticus DNA polymerase I mutants with altered fidelity. Interaction mutations in the O-Helix.J Biol Chem 275:32728-32735), higher DNA polymerization activity (Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering. Front Microbiol 5:461. Doi:10.3389/fmicb.2014.00461), high concentration inhibitor tolerance (Zhang Z, kermekchiev MB, barnes WM (2010) Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq.J. Dign 12:152-161), reduced 5'-3' exonuclease activity (Vains htein I, atrazhev A, eom SH, elliott JF, wistar DS, malcolm BA (1996) Peptide rescue of an N-Terminal truncation of the Stoffel fragment of Taq DNA mease.Protein 5:51785-51792).

The modification of Taq enzyme mainly comprises the following ways. 1: the domain is added to give it new properties. For example, a single-chain binding domain (SSB) or a DNA binding protein Sso7 is added, so that the binding capacity of Taq enzyme to primers and template DNA is enhanced, and the primer and template DNA has stronger extension capacity and continuous synthesis capacity, and is suitable for amplification reaction of long-fragment DNA. However, increasing the domain directly increases the molecular weight of Taq enzyme, which may decrease the solubility and stability of Taq enzyme. The yield of prokaryotic expression production is reduced. 2: unnecessary domains on Taq enzyme are removed. If the 5'-3' exonuclease domain (the first 280 amino acids of the N end of Taq enzyme) is deleted, the Taq enzyme only keeps the active region of nucleic acid polymerase, the possibility of degrading primer and template DNA by high-concentration Taq enzyme is reduced, and the aim of improving the polymerization activity of Taq enzyme is fulfilled. However, the Taq enzyme mutant obtained by the method does not have 5'-3' exonuclease activity, is not suitable for quantitative PCR reaction based on Taq man probe method, and has limited application range. 3: site-directed mutagenesis. Site-directed mutagenesis of the amino acids of the active site, magnesium ion binding site, and DNA binding site is performed to increase the affinity of each site for substrates, templates, and primers, thereby increasing tolerance to various inhibitors. Because of the complexity of the protein structure, some amino acids far from the active site may also affect the overall structure of the enzyme, so that it is difficult to modify the enzyme as a whole by only mutating amino acids at specific active sites. Moreover, the existing computer simulation technology is difficult to predict what influence each site mutation has on the whole structure, the workload of preparing mutants by using a site-directed mutagenesis method and screening the mutants is very large, the efficiency is low, and some sites which possibly have great influence on the activity cannot be identified.

Disclosure of Invention

The present invention aims to provide a thermostable DNA polymerase mutant having high amplification activity.

In a first aspect of the invention, there is provided a mutant DNA polymerase, which mutant DNA polymerase has a mutation at one or more sites selected from the group consisting of: v453, F495, E507, K508, T509, a518, S624, Y672, E734, R737, F749, T757, L764, H785, wherein the amino acid residue numbering uses that shown in SEQ ID No. 2.

In another preferred embodiment, the mutant DNA polymerase has at least 1.5 times the activity of a wild-type DNA polymerase (SEQ ID NO.: 2); preferably at least 2 times; more preferably at least 3 times.

In another preferred embodiment, the amino acid sequence of the wild-type DNA polymerase is set forth in SEQ ID NO. 2.

In another preferred embodiment, the amino selected sequence of the mutant DNA polymerase has at least 80% homology to SEQ ID No. 2; more preferably, it has a homology of at least 90%; most preferably, having at least 95% homology; such as having at least 96%, 97%, 98%, 99% homology.

In another preferred embodiment, the mutant DNA polymerase is selected from the group consisting of mutants 1-20 of:

mutant numbering	Mutant amino acids
		1	E507A，K508L，E734E，F749K
2	K508L、V453A、R737K
		3	E734G
4	F749G K508L L764K
		5	E507Q、T757S
6	H785G
		7	S624T F749V
8	E734F F749V
		9	K508L R737W Y672R
10	E507H H785L
		11	A518Q E734M
12	F495R F749T
		13	K508L F749T E734F
14	R737P S624K
		15	T757W V453G E507M
16	F749E H785G F495G
		17	E734F Y672P
18	T509L H785K
		19	E734G T757S L764Q
20	K508L V453A A518Q。

In another preferred embodiment, the number of mutation sites in the mutated DNA polymerase is 1-4, preferably 2 or 3.

In another preferred embodiment, the mutant DNA polymerase is selected from each of the specific mutant enzymes in table 2.

In another preferred embodiment, the mutant DNA polymerase comprises the mutation site of each particular mutant enzyme in table 2.

In another preferred embodiment, the mutant DNA polymerase is mutated on the basis of the wild-type DNA polymerase shown in SEQ ID NO.2, and the mutant DNA polymerase comprises a mutation site selected from the group consisting of:

(1)E507A、K508L、E734E、F749K；

(2)K508L、V453A、R737K

(3)E734G

(4)F749G、K508L、L764K

(5)E507Q、T757S

(6)H785G

(7)S624T、F749V

(8)E734F、F749V

(9)K508L、R737W、Y672R

(10)E507H、H785L

(11)A518Q、E734M

(12)F495R、F749T

(13)K508L、F749T、E734F

(14)R737P、S624K

(15)T757W、V453G、E507M

(16)F749E、H785G、F495G

(17)E734F、Y672P

(18)T509L、H785K

(19) E734G, T757S, L764Q; and

(20)K508L、V453A、A518Q。

in a second aspect of the invention there is provided a polynucleotide molecule encoding a mutated DNA polymerase according to the first aspect of the invention.

In a third aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In a fourth aspect of the invention there is provided a host cell comprising a vector or chromosome according to the first aspect of the invention incorporating a nucleic acid molecule according to the second aspect of the invention.

In another preferred embodiment, the host cell is a prokaryotic cell, or a eukaryotic cell.

In another preferred embodiment, the prokaryotic cell is E.coli.

In another preferred embodiment, the eukaryotic cell is a yeast cell.

In a fifth aspect of the invention, there is provided a method of preparing a mutant DNA polymerase according to the first aspect of the invention, comprising the steps of:

(i) Culturing the host cell of the fourth aspect of the invention under suitable conditions to express the mutant DNA polymerase; and

(ii) Isolating said mutated DNA polymerase.

In another preferred embodiment, the temperature at which the host cells are cultured in step (i) is from 20℃to 40 ℃; preferably from 25℃to 37℃such as 35 ℃.

In a sixth aspect of the invention there is provided a kit comprising a mutated DNA polymerase according to the first aspect of the invention.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Detailed Description

Through extensive and intensive research, the inventor builds a random mutation library for a polymerase activity structural domain of Taq enzyme by applying a protein directed evolution technology, naturally eliminates unsuitable mutation by gradually adding screening pressure, gradually accumulates mutation with dominant character, finally screens out a series of amino acid sites and mutation thereof which have key effects on the amplification performance and the polymerization performance of the Taq enzyme, and obtains Taq enzyme mutants with high amplification performance. On this basis, the present invention has been completed.

Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

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. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

Taq enzyme

Taq enzyme is widely used in Polymerase Chain Reaction (PCR), and is the first enzyme for nucleic acid amplification, detection and other reactions. Commercial Taq enzyme was cloned and expressed using E.coli prokaryotic expression system.

The wild type Taq enzyme DNA sequence is as follows:

ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACGCGTTCTTCTTGTGGATGGACATCATCTGGCGTACCGTACCTTTCATGCCCTGAAGGGCCTGACCACTTCGCGTGGGGAACCCGTGCAAGCAGTTTATGGATTCGCCAAATCGTTACTTAAGGCTCTGAAGGAGGATGGTGATGCGGTCATTGTTGTGTTCGACGCAAAAGCTCCCTCGTTCCGTCACGAGGCCTACGGCGGCTATAAAGCTGGGCGTGCACCCACACCTGAGGATTTTCCCCGGCAACTTGCTTTGATAAAGGAATTAGTAGACCTGTTAGGCCTGGCGCGGTTAGAAGTGCCGGGTTACGAAGCAGATGACGTCTTGGCTAGTTTAGCGAAAAAGGCTGAAAAAGAGGGATATGAAGTGCGGATCCTGACCGCGGATAAAGATCTGTATCAACTGTTGTCCGACCGTATTCACGTGCTTCATCCGGAGGGCTACTTGATAACCCCGGCTTGGCTGTGGGAGAAATATGGGCTGCGTCCAGATCAGTGGGCTGATTATCGTGCACTTACAGGCGATGAATCTGATAATCTTCCCGGCGTCAAGGGGATTGGTGAGAAAACCGCCCGTAAACTTTTGGAGGAGTGGGGCAGCTTGGAGGCGCTGTTGAAGAATCTGGATCGTTTGAAACCCGCTATACGGGAAAAAATCTTGGCGCACATGGACGACTTAAAACTGTCTTGGGACCTGGCGAAAGTTCGTACTGATTTGCCGCTGGAGGTCGACTTTGCGAAGCGTCGCGAGCCCGATCGTGAACGTCTTCGCGCATTTCTGGAGCGTTTAGAATTTGGCTCCCTGTTGCATGAGTTTGGTTTGCTTGAAAGCCCGAAGGCACTTGAGGAAGCTCCTTGGCCTCCGCCTGAGGGCGCTTTTGTCGGATTTGTCTTGAGCCGTAAAGAACCGATGTGGGCGGACTTACTGGCCCTTGCTGCTGCTCGTGGGGGTCGCGTGCATCGCGCACCGGAGCCATACAAAGCACTTCGTGACCTTAAAGAAGCCCGTGGCTTGTTGGCAAAAGATTTAAGTGTCCTGGCTTTACGCGAGGGCTTGGGCTTACCACCGGGAGATGATCCGATGCTTTTGGCCTATCTGCTGGACCCGAGCAACACGACTCCAGAGGGCGTTGCCCGTCGTTATGGCGGAGAATGGACGGAGGAGGCGGGAGAGCGCGCAGCGTTAAGCGAGCGTCTGTTTGCTAATCTGTGGGGACGCTTAGAGGGAGAGGAGCGCCTGTTGTGGTTGTACCGTGAAGTGGAACGGCCGCTGAGTGCAGTGTTAGCTCACATGGAAGCAACCGGGGTGCGGCTGGACGTTGCGTATTTGCGTGCGCTGTCGTTAGAGGTCGCGGAGGAAATAGCCCGTCTGGAGGCCGAAGTATTCCGTTTGGCTGGCCATCCTTTCAACCTGAACAGTCGGGATCAGCTGGAACGTGTACTTTTTGATGAACTGGGGCTGCCCGCCATCGGCAAAACCGAAAAAACCGGCAAACGTAGCACCTCTGCGGCAGTGCTGGAAGCGTTACGTGAAGCTCATCCGATTGTGGAGAAAATTCTGCAATATCGCGAATTGACGAAACTGAAGAGCACCTATATTGATCCGCTGCCAGACTTAATTCACCCCCGTACCGGACGGTTGCATACCCGCTTCAACCAGACCGCGACGGCGACAGGGCGGCTGAGTAGCAGCGATCCGAACCTGCAAAACATTCCCGTGCGTACCCCGCTGGGTCAGCGTATTCGCCGTGCTTTCATTGCCGAGGAAGGCTGGCTGCTGGTCGCGCTGGACTACTCGCAAATCGAATTGCGTGTGTTGGCCCACCTGTCGGGCGACGAAAACTTAATACGCGTGTTTCAAGAAGGTCGTGACATACATACTGAAACCGCGTCCTGGATGTTTGGAGTCCCACGGGAGGCTGTCGATCCTCTTATGCGTCGTGCCGCCAAAACAATTAACTTCGGAGTTCTGTACGGCATGTCGGCACATCGTTTATCACAGGAACTGGCGATTCCGTATGAAGAAGCGCAGGCCTTCATAGAACGTTATTTCCAATCATTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAAGAGGGCCGTCGTCGTGGCTATGTAGAGACTCTGTTCGGACGTCGGCGGTATGTACCCGATCTTGAGGCCCGTGTGAAGTCCGTTCGTGAGGCAGCAGAACGTATGGCGTTTAACATGCCAGTCCAGGGCACAGCGGCGGACCTGATGAAATTAGCTATGGTTAAGCTGTTTCCGCGTTTGGAAGAAATGGGCGCTCGTATGCTGTTACAGGTTCATGACGAGTTAGTATTAGAAGCACCGAAGGAGCGTGCCGAAGCCGTGGCCCGGTTAGCCAAAGAGGTAATGGAAGGCGTCTACCCCCTTGCAGTCCCGCTTGAAGTCGAAGTTGGCATAGGGGAAGACTGGTTATCTGCGAAGGAA(SEQ ID NO.:1)

the amino acid sequence of the wild Taq enzyme is as follows:

MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE(SEQ ID NO.:2)

the invention screens out amino acid sites and mutation modes thereof which are highly relevant to the amplification activity of Taq enzyme by a directed evolution mode. Related mutated amino acid positions include: v453, F495, E507, K508, T509, a518, S624, Y672, E734, R737, F749, T757, L764, and H785, amino acid residue numbering according to SEQ ID No. 2. Mutation of the amino acid site into any other amino acid can obtain higher activity Taq enzyme mutant, and preferred mutant forms include: E507A/Q/H/M, K508L, E734G/F/M, F749K/G/V/T/E, L764K/Q, V453A/G, R737K/W/P, T757S/W, H785G/L/K, S624T/K, Y672R/P, A518Q, F G/R, T509L.

The invention screens out amino acid sites with high correlation to Taq enzyme activity and mutation modes thereof from a random mutation library by directed evolution technology, wherein the number of mutants is 10 of site-directed mutation ⁵ Multiple times, it is more advantageous to screen mutant sites with synergistic effects, which cannot be predicted by existing computer modeling techniques. Based on the principle of directed evolution, the accumulated dominant traits are most suitable for the added screening conditions, so that the obtained mutants are definitely all the mutationsOptimal individuals in the body.

The Taq enzyme gene sequences of the present invention may be obtained by conventional methods, such as total artificial synthesis or PCR synthesis, which are available to those of ordinary skill in the art. One preferred synthesis method is an asymmetric PCR method. The asymmetric PCR method is to amplify a large amount of single-stranded DNA (ssDNA) by PCR using a pair of primers in unequal amounts. The pair of primers is referred to as non-limiting primer and limiting primer, respectively, in a ratio of typically 50-100:1. During the first 10-15 cycles of the PCR reaction, the amplified product is mainly double stranded DNA, but when the restriction primer (low concentration primer) is consumed, the non-restriction primer (high concentration primer) directed PCR will produce a large amount of single stranded DNA. Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The Taq enzyme of the present invention may be expressed or produced by conventional recombinant DNA techniques comprising the steps of:

(1) Transforming or transducing a suitable host cell with a polynucleotide encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) Culturing the host cell in a suitable medium;

(3) The target protein is separated and purified from the culture medium or cells, thereby obtaining Taq enzyme.

Methods well known to those skilled in the art can be used to construct expression vectors comprising the coding DNA sequences of the Taq enzyme of the invention and appropriate transcriptional/translational control signals, preferably commercially available vectors: pET28. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In addition, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

The recombinant vector comprises in the 5 'to 3' direction: a promoter, a gene of interest and a terminator. If desired, the recombinant vector may further comprise the following elements: a protein purification tag; a 3' polynucleotide acidification signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; selection markers (antibiotic resistance genes, fluorescent proteins, etc.); an enhancer; or an operator.

Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid or vector may be used as long as it is capable of replication and stability in a host.

The person skilled in the art can construct vectors containing the promoter and/or the gene sequence of interest of the present invention by means of well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The expression vectors of the invention may be used to transform an appropriate host cell to allow the host to transcribe the RNA of interest or to express the protein of interest. The host cell may be a prokaryotic cell such as E.coli, corynebacterium glutamicum, brevibacterium flavum, streptomyces, agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., E.coli), caCl may be used ₂ The treatment can also be carried out by electroporation. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The transformed plant may also be transformed by Agrobacterium or gene gun, such as leaf disc method, embryo transformation method, flower bud soaking method, etc. Plants can be regenerated from the transformed plant cells, tissues or organs by conventional methods to obtain transgenic plants.

The term "operably linked" refers to the attachment of a gene of interest to be expressed by transcription to its control sequences in a manner conventional in the art.

Culturing engineering bacteria and fermenting production of target protein

After obtaining the engineered cells, the engineered cells may be cultured under appropriate conditions to express the protein encoded by the gene sequence of the present invention. The medium used in the culture may be selected from various conventional media according to the host cell, and the culture is performed under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

In the present invention, conventional fermentation conditions may be employed. Representative conditions include (but are not limited to):

(a) In terms of temperature, the fermentation and induction temperatures of Taq enzyme are kept at 25-37 ℃;

(b) The pH value in the induction period is controlled to be 3-9;

(c) In the case of Dissolved Oxygen (DO), the DO is controlled to be 10-90%, and the maintenance of dissolved oxygen can be solved by the introduction of oxygen/air mixed gas;

(d) For the feeding, the type of the feeding preferably comprises carbon sources such as glycerol, methanol, glucose and the like, and the feeding can be carried out independently or by mixing;

(e) As for the induction period IPTG concentration, conventional induction concentrations can be used in the present invention, and usually the IPTG concentration is controlled to 0.1-1.5mM;

(f) The induction time is not particularly limited, and is usually 2 to 20 hours, preferably 5 to 15 hours.

The target protein Taq enzyme exists in the cell of the escherichia coli, host cells are collected by a centrifugal machine, then the host cells are crushed by high pressure, mechanical force, enzymolysis cell cover or other cell crushing methods, and recombinant proteins are released, preferably a high pressure method. The host cell lysate can be purified primarily by flocculation, salting out, ultrafiltration and other methods, and then subjected to chromatography, ultrafiltration and other purification methods, or can be directly subjected to chromatography purification.

The chromatographic techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc. Common chromatographic methods include:

1. anion exchange chromatography:

anion exchange chromatography media include (but are not limited to): Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermentation sample is high, which affects the binding to the ion exchange medium, the salt concentration is reduced before ion exchange chromatography is performed. The sample can be replaced by dilution, ultrafiltration, dialysis, gel filtration chromatography and other means until the sample is similar to the corresponding ion exchange column equilibrium liquid system, and then the sample is loaded to perform gradient elution of salt concentration or pH.

2. Hydrophobic chromatography:

hydrophobic chromatography media include (but are not limited to): phenyl-Sepharose, butyl-Sepharose, octyle-Sepharose. Sample by adding NaCl, (NH) ₄ ) ₂ SO ₄ And the salt concentration is increased in an equal mode, then the sample is loaded, and the sample is eluted by a method of reducing the salt concentration. The hetero proteins with a large difference in hydrophobicity were removed by hydrophobic chromatography.

3. Gel filtration chromatography

Hydrophobic chromatography media include (but are not limited to): sephacryl, superdex, sephadex. The buffer system is replaced by gel filtration chromatography or further purified.

4. Affinity chromatography

Affinity chromatography media include (but are not limited to): hiTrap ^TM HeparinHPColumns。

5. Membrane filtration

The ultrafiltration medium comprises: organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, and metal membranes. The purposes of purification and concentration can be achieved by membrane filtration.

The invention has the main advantages that:

(1) The heat-resistant DNA polymerase mutant with high amplification activity has obviously improved product quantity obtained by amplification under the same PCR cycle times compared with wild Taq enzyme.

(2) The time required for amplifying and producing the same amount of products under the same condition by the heat-resistant DNA polymerase mutant with high amplification activity is obviously shortened compared with that of wild type Taq enzyme, so that the detection efficiency can be obviously improved.

The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Example 1: construction of Taq enzyme random mutant plasmid

Amplifying the polymerase activity structural domain DNA sequence (423-831 amino acid coding sequence) of the Taq enzyme by using low-fidelity PCR (Error-PCR), wherein the mutation occurrence rate is 0.3%, then connecting with the rest coding sequences (1-423 amino acid sequences) of the Taq enzyme, and cloning into a pET28a prokaryotic expression vector to obtain the Taq enzyme random mutation plasmid. The method comprises the following specific steps:

1) The Taq-pET28a plasmid is used as a template, and a primer T (1-423) is designed to amplify the Taq (1-423) fragment.

Taq(1-423)DNA Seq

ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACGCGTTCTTCTTGTGGATGGACATCATCTGGCGTACCGTACCTTTCATGCCCTGAAGGGCCTGACCACTTCGCGTGGGGAACCCGTGCAAGCAGTTTATGGATTCGCCAAATCGTTACTTAAGGCTCTGAAGGAGGATGGTGATGCGGTCATTGTTGTGTTCGACGCAAAAGCTCCCTCGTTCCGTCACGAGGCCTACGGCGGCTATAAAGCTGGGCGTGCACCCACACCTGAGGATTTTCCCCGGCAACTTGCTTTGATAAAGGAATTAGTAGACCTGTTAGGCCTGGCGCGGTTAGAAGTGCCGGGTTACGAAGCAGATGACGTCTTGGCTAGTTTAGCGAAAAAGGCTGAAAAAGAGGGATATGAAGTGCGGATCCTGACCGCGGATAAAGATCTGTATCAACTGTTGTCCGACCGTATTCACGTGCTTCATCCGGAGGGCTACTTGATAACCCCGGCTTGGCTGTGGGAGAAATATGGGCTGCGTCCAGATCAGTGGGCTGATTATCGTGCACTTACAGGCGATGAATCTGATAATCTTCCCGGCGTCAAGGGGATTGGTGAGAAAACCGCCCGTAAACTTTTGGAGGAGTGGGGCAGCTTGGAGGCGCTGTTGAAGAATCTGGATCGTTTGAAACCCGCTATACGGGAAAAAATCTTGGCGCACATGGACGACTTAAAACTGTCTTGGGACCTGGCGAAAGTTCGTACTGATTTGCCGCTGGAGGTCGACTTTGCGAAGCGTCGCGAGCCCGATCGTGAACGTCTTCGCGCATTTCTGGAGCGTTTAGAATTTGGCTCCCTGTTGCATGAGTTTGGTTTGCTTGAAAGCCCGAAGGCACTTGAGGAAGCTCCTTGGCCTCCGCCTGAGGGCGCTTTTGTCGGATTTGTCTTGAGCCGTAAAGAACCGATGTGGGCGGACTTACTGGCCCTTGCTGCTGCTCGTGGGGGTCGCGTGCATCGCGCACCGGAGCCATACAAAGCACTTCGTGACCTTAAAGAAGCCCGTGGCTTGTTGGCAAAAGATTTAAGTGTCCTGGCTTTACGCGAGGGCTTGGGCTTACCACCGGGAGATGATCCGATGCTTTTGGCCTATCTGCTGGACCCGAGCAACACGACTCCAGAGGGCGTTGCCCGTCGTTATGGCGGAGAATGGACGGAGGAGGCGGGAGAGCGCGCAGCGTTAAGCGAGCGTCTGTTTGCTAATCTGTGGGGACGCTTAGAGGGAGAG(SEQ ID NO.:3)

T1-423_PF：5'ATATCATATGCGTGGCATGCTGCCGCTTTT 3'(SEQ ID NO.:4)

T1-423_PR：5'GCATGAATTCCGTCTCCTCTCCCTCTAAGC 3'(SEQ ID NO.:5)

PCR reaction system and procedure:

PCR procedure: 95℃for 3 minutes, (95℃for 30 seconds, 60℃for 30 seconds, 72℃for 1 minute). Times.25 cycles, 72℃for 3 minutes, 4℃for preservation

The PCR product was recovered by purification using a DNA gel recovery kit, digested with NdeI and XhoI, ligated into pET28a vector, and sequenced to confirm correct sequence, and the resulting plasmid was designated Taq (1-423) -pET28

2) Using the Taq-pET28a plasmid as a template, clontech was usedPCR Random Mutagenesis Kit (Dalianbao organism PT 3393-2), the primer (TMu _F/R) is designed to amplify Taq (423-822) fragment

Taq(423-832)DNA Seq

GGAGAGGAGCGCCTGTTGTGGTTGTACCGTGAAGTGGAACGGCCGCTGAGTGCAGTGTTAGCTCACATGGAAGCAACCGGGGTGCGGCTGGACGTTGCGTATTTGCGTGCGCTGTCGTTAGAGGTCGCGGAGGAAATAGCCCGTCTGGAGGCCGAAGTATTCCGTTTGGCTGGCCATCCTTTCAACCTGAACAGTCGGGATCAGCTGGAACGTGTACTTTTTGATGAACTGGGGCTGCCCGCCATCGGCAAAACCGAAAAAACCGGCAAACGTAGCACCTCTGCGGCAGTGCTGGAAGCGTTACGTGAAGCTCATCCGATTGTGGAGAAAATTCTGCAATATCGCGAATTGACGAAACTGAAGAGCACCTATATTGATCCGCTGCCAGACTTAATTCACCCCCGTACCGGACGGTTGCATACCCGCTTCAACCAGACCGCGACGGCGACAGGGCGGCTGAGTAGCAGCGATCCGAACCTGCAAAACATTCCCGTGCGTACCCCGCTGGGTCAGCGTATTCGCCGTGCTTTCATTGCCGAGGAAGGCTGGCTGCTGGTCGCGCTGGACTACTCGCAAATCGAATTGCGTGTGTTGGCCCACCTGTCGGGCGACGAAAACTTAATACGCGTGTTTCAAGAAGGTCGTGACATACATACTGAAACCGCGTCCTGGATGTTTGGAGTCCCACGGGAGGCTGTCGATCCTCTTATGCGTCGTGCCGCCAAAACAATTAACTTCGGAGTTCTGTACGGCATGTCGGCACATCGTTTATCACAGGAACTGGCGATTCCGTATGAAGAAGCGCAGGCCTTCATAGAACGTTATTTCCAATCATTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAAGAGGGCCGTCGTCGTGGCTATGTAGAGACTCTGTTCGGACGTCGGCGGTATGTACCCGATCTTGAGGCCCGTGTGAAGTCCGTTCGTGAGGCAGCAGAACGTATGGCGTTTAACATGCCAGTCCAGGGCACAGCGGCGGACCTGATGAAATTAGCTATGGTTAAGCTGTTTCCGCGTTTGGAAGAAATGGGCGCTCGTATGCTGTTACAGGTTCATGACGAGTTAGTATTAGAAGCACCGAAGGAGCGTGCCGAAGCCGTGGCCCGGTTAGCCAAAGAGGTAATGGAAGGCGTCTACCCCCTTGCAGTCCCGCTTGAAGTCGAAGTTGGCATAGGGGAAGACTGGTTATCTGCGAAGGAATAA(SEQ ID NO.:6)

TMu_F：5'GGAGAGGAGCGCCTGTTGTGGTTGT 3'(SEQ ID NO.:7)

TMu_R：5'TTATTCCTTCGCAGATAACCAGTCT 3'(SEQ ID NO.:8)

PCR reaction system and procedure:

x25 cycles at 95℃for 3 minutes, (95℃for 30 seconds, 60℃for 30 seconds, 68℃for 2 minutes), 68℃for 5 minutes, 4℃for preservation

The PCR product was digested with BsmBI and XhoI, followed by ligation of BsmBI and XhoI digested Taq (1-423) -pET28 plasmids, transformation of BL21 (DE 3) expression host bacteria with the ligation product, and counting of transformants.

Example 2: expression and directed evolution screening of Taq enzyme mutants

The Taq enzyme mutant plasmid is transformed into BL21 (DE 3) expression strain, and Taq enzyme mutant library is induced and expressed. BL21 (DE 3) induced expression bacteria containing Taq enzyme mutant library are dispersed and wrapped by an emulsion PCR system, and PCR reaction is carried out to amplify DNA containing Taq enzyme mutant fragments. And then, carrying out high-fidelity PCR secondary amplification on the DNA fragment amplified by emulsion PCR by using a Taq enzyme specific primer, and re-cloning the amplified DNA product into a pET28a expression vector to complete a primary screening process. And then repeating the screening process of emulsion PCR-secondary high-fidelity PCR-cloning to the pET28a expression vector, and simultaneously gradually shortening the extension time of emulsion PCR in each screening so as to accumulate mutant populations with high extension activity and high amplification activity. The method comprises the following specific steps:

1) The transformant obtained in example 1 was inoculated into LB medium, shake-cultured at 37℃for 6 hours, and then added with isopropyl thiogalactoside (IPTG) at a final concentration of 0.1mM, followed by induction culture at 37℃for 3 hours. The cells were collected by centrifugation and subjected to ddH ₂ Washing the cells twice with O and finally with ddH ₂ O-resuspension of the cells, determination of the absorbance of the cell solution at 600nm (OD 600 value), dilution of the final concentration with ddH2O to od600=1.0

2) Preparing an oil phase solution

Tween-80 200ul

Triton X-100 25ul

Mineral oil 10ml

Mixing the above 3 reagents, and mixing

3) Preparation of aqueous phase reaction solution

Diluting the bacterial body weight suspension prepared in step 1) with ddH2O 100 times to prepare the following reaction solution

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pET28_F primer: TACGGTTAACCCTTTGAATCA (SEQ ID NO.: 9)

pET28_R primer: GTTACCTGGTTAAACTGTACT (SEQ ID NO.: 10)

4) Preparation of emulsion systems

200ul of water phase and 400ul of oil phase are mixed in a 2ml tube, the mixture is oscillated for 10 minutes on a vortex oscillator at high speed, 5 PCR tubes are taken, 100ul of mixed solution is respectively packaged, and the procedure is as follows: 95℃for 5 minutes, (95℃for 30 seconds, 55℃for 30 seconds, 72℃for 2 minutes) X25 cycles, 72℃for 5 minutes, 4℃for infinity

5) The emulsion PCR product was transferred to a 1.5ml tube, 166ul of water saturated diethyl ether was added, vortexed for 30 seconds, centrifuged at 12000rpm for 10 minutes, the lower liquid phase was transferred away, left to stand at room temperature for 10 minutes until diethyl ether volatilized, the liquid phase product was purified by phenol chloroform extraction, and ethanol precipitation was performed overnight to recover the product.

6) High-fidelity PCR secondary amplification product

Performing PCR secondary amplification by taking the product of the step 4) as a template

The PCR procedure was as follows: 95℃for 5 minutes, 20 cycles X (95℃for 30 seconds, 62℃for 30 seconds, 72℃for 2 minutes) at 72℃for 5 minutes, 4℃infinity

Taq_F primer: ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACG (SEQ ID NO.: 11)

Taq_R primer: TTCCTTCGCAGATAACCAGTCTTCCCCTATGCCAACTTCGAC (SEQ ID NO.: 12)

7) The PCR product was purified using a DNA product purification recovery kit and then religated with the pET28a expression vector. Thus far complete a round of screening

8) Repeating steps (1) - (6) with the transformant re-ligated to pET28a vector, changing the conditions of emulsion PCR according to the procedure of the following table, and gradually adding selection pressure to the mutant pool

Second round screening: 95℃for 5 minutes, (95℃for 30 seconds, 55℃for 30 seconds, 72℃for 1.5 minutes) X25 cycles, 72℃for 5 minutes, 4℃for infinity

Third round screening: 95℃for 5 minutes, (95℃for 30 seconds, 55℃for 30 seconds, 72℃for 1 minute) X20 cycles, 72℃for 5 minutes, 4℃for infinity

Fourth screening: 95℃for 5 minutes, (95℃for 30 seconds, 55℃for 30 seconds, 72℃for 30 seconds) X15 cycles, 72℃for 5 minutes, 4℃infinity

After 4 rounds of screening, the resulting Taq enzyme mutant transformants were subjected to high throughput screening of example 3

Example 3: high throughput screening of Taq enzyme mutants

384 single clones were randomly selected from the mutant library obtained in example 2, cultured and induced for expression, and their amplification activities were tested by high-throughput PCR reaction, from which 20 mutants with high amplification activities were selected. The method comprises the following specific steps:

1) 384 single clones were selected, inoculated into LB medium, cultured at 37℃for 6 hours, added with isopropyl thiogalactoside (IPTG) at a final concentration of 0.1mM, and induced at 37℃for 3 hours

2) After the induction culture, the cells were collected by centrifugation, a lysate (50Mm Tris,50Mm NaCl,5% glycerol pH 8.5) containing 0.1mg/ml lysozyme was added, and the cells were resuspended and incubated at 37℃for 10 minutes and heated at 75℃for 30 minutes. Then, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was collected.

3) Taking 96-well PCR plate, adding the following reaction components into each well

PCR procedure: 95℃for 5 minutes, 20 cycles X (95℃for 30 seconds, 62℃for 30 seconds, 72℃for 60 seconds), 4C-infinity

And 5ul of PCR products are taken for agarose gel electrophoresis, the yield of the PCR products of the supernatants prepared by each monoclonal is compared, and the monoclonal with the highest yield is selected. The amplification yield of each mutant was 1.2-fold and 2-fold that of the wild-type.

Example 4: confirmation of mutation sites of dominant Taq enzyme mutant

DNA sequence sequencing was performed on the Taq enzyme mutants selected in example 3, and the mutation condition of the amino acid sequence was determined, and high frequency mutation sites and mutation forms thereof were counted.

TABLE 1

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Sequencing the 20 mutants with better amplification activity, and counting the amino acid mutation conditions of the mutants as shown in the table above, wherein the results show that: v453, F495, E507, K508, T509, A518, S624, Y672, E734, R737, F749, T757, L764, H785 were repeated at 20 mutant high frequencies, demonstrating that the mutation has a significant effect on the amplification activity of Taq enzyme.

EXAMPLE 5 comparison of mutant modified Taq enzyme with wild-type Taq enzyme

Taking Taq enzyme mutant 1, carrying out the following amplification capability test with wild Taq enzyme after expression and purification:

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PCR procedure: 95℃for 5 minutes, n cycles X (95℃for 15 seconds, 55℃for 15 seconds, 72℃for 10 seconds), 4C-infinity

Preparing the reaction liquid, carrying out PCR amplification for 15, 20, 25 and 30 cycles, precipitating and purifying the PCR product by ethanol, measuring the light absorption value of the product at 260nm, and calculating the total amount (ng) of the PCR product corresponding to each cycle number. The results were as follows:

TABLE 2

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From the results, the amplification of Taq enzyme mutants 1 to 20 at the same PCR cycle number can significantly improve the amount of the amplified product compared with the wild Taq enzyme. Wherein, the product quantity obtained when the mutant 1 is amplified for 20 cycles is already equivalent to the product quantity obtained when the wild type Taq enzyme is amplified for 30 cycles; under the condition of 30 amplification cycles, the obtained product quantity of the mutant 1 is more than 2.5 times of that of wild Taq enzyme.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A mutant DNA polymerase, wherein the mutant DNA polymerase is mutated based on the wild-type DNA polymerase shown in SEQ ID No.2, and wherein the mutation is: mutation of the 624 th amino acid residue to T and mutation of the 749 th amino acid residue to V.

2. The DNA polymerase of claim 1, wherein the mutant DNA polymerase has at least 1.2 times the activity of a wild-type DNA polymerase; preferably at least 1.5 times.

3. A polynucleotide molecule encoding the mutant DNA polymerase of claim 1.

4. A vector comprising the polynucleotide molecule of claim 3.

5. A host cell comprising the vector of claim 4 or a chromosome incorporating the polynucleotide molecule of claim 3.

6. The host cell of claim 5, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

7. The host cell of claim 6, wherein the prokaryotic cell is an e.

8. The host cell of claim 6, wherein the eukaryotic cell is a yeast cell.

9. A method of preparing the mutant DNA polymerase of claim 1, comprising the steps of:

(i) Culturing the host cell of claim 5 under suitable conditions to express said mutated DNA polymerase; and

(ii) Isolating said mutated DNA polymerase.

10. A kit comprising the mutated DNA polymerase of claim 1.