A kind of high processivity Pfu DNA polymerase and its preparation method and application

Abstract

The present invention discloses a highly progressive Pfu DNA polymerase and a preparation method and application thereof, wherein the highly progressive Pfu DNA polymerase is specifically: a mutant dCE expressing colicin CE is fused to the N-terminus of a wild-type Pfu DNA polymerase protein sequence, wherein the gene sequence of dCE is shown in any one of SEQ ID NO.2‑5. Compared with the wild-type Pfu DNA polymerase, the amplification speed of the modified polymerase can reach about 8 kb / min, which is much higher than the wild-type Pfu DNA polymerase, and the amplification yield is also significantly improved. Therefore, the highly progressive Pfu DNA polymerase provided by the present invention has broad application prospects in gene cloning, high-fidelity amplification of long fragments, and PCR diagnosis.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and protein engineering, and specifically relates to a highly processive PfuDNA polymerase and a preparation method and application thereof. Background Art

[0002] In 1986, American Mullis invented the polymerase chain reaction (PCR), which can efficiently amplify specific DNA fragments in vitro. During the PCR process, double-stranded DNA will denature into single strands at high temperatures; during the cooling process (generally around 60°C), the single-stranded primer follows the principle of base complementary pairing and binds to the corresponding region of the single-stranded DNA template; then the DNA polymerase synthesizes the complementary chain in the 5'-3' direction under medium temperature conditions. With the application of Taq DNA polymerase in PCR, PCR technology has gotten rid of the dilemma of needing to supplement enzymes in each cycle and ushered in a golden period of development. Thanks to the high efficiency and high specificity of PCR technology, this technology has been widely used in life science and medical fields such as molecular cloning, gene mutagenesis, sequencing, and molecular diagnosis.

[0003] At present, the commonly used DNA polymerases in PCR generally have the problems of low amplification efficiency and insufficient fidelity. For example, TaqDNA polymerase, as the most widely used type of DNA polymerase in PCR, has an amplification speed of about 1kb / min, which is much lower than the DNA synthesis speed in vivo. Moreover, the enzyme lacks 3'-5' exonuclease activity, has poor amplification fidelity, and can amplify relatively short fragments. Compared with Taq DNA polymerase, Pfu DNA polymerase from the extreme thermophilic bacterium Pyrococcusfuriosus COM1 has 5'-3' DNA polymerase activity and 3'-5' exonuclease activity, and its fidelity is 12 times that of Taq DNA polymerase, with an error rate of about 1.4±0.2×10 -6 . However, previous studies have shown that the extension rate of Pfu DNA polymerase is lower than that of Taq DNA polymerase, about 600bp / min. In order to improve the amplification efficiency of Pfu DNA polymerase, researchers have tried to modify the enzyme. In 2004, Wang et al. fused Sso7d from Sulfolobus solfataricus with Pfu DNA polymerase to express the fusion protein Phusion, and the amplification rate was increased to 2-4kb / min. At the same time, its fidelity was greatly improved, 50 times higher than Taq DNA polymerase and 6 times higher than Pfu DNA polymerase. The enzyme has been commercialized by Thermofisher.

[0004] The colicins produced by Escherichia coli are a class of protein toxins that can kill closely related bacilli. The DNA enzymes of the colicin E family include CE2, CE7, CE8 and CE9, which are a class of non-specific nucleases. Reports have shown that the structures of the four CEs are similar, and their DNA enzyme domains are located at the C-terminus of the protein molecule, about 15 kDa. The truncated C-terminal domain has DNA binding and cleavage capabilities. In further experiments, the dCE mutants obtained by mutating the amino acids involved in DNA cleavage in the C-terminal domains of the four CEs still retain strong DNA binding activity, but do not cleave DNA. Summary of the invention

[0005] In the previous work, the inventors found that dCE has good thermal stability, so they tried to fuse these four dCE mutants (dCE2, dCE7, dCE8 and dCE9) with Pfu DNA polymerase for expression, in order to improve the enzymatic properties of Pfu DNA polymerase through the binding activity of dCE with DNA.

[0006] The specific technical scheme of the present invention is as follows:

[0007] The first aspect of the present invention provides a method for improving the processivity of Pfu DNA polymerase, specifically: by fusing and expressing the mutant dCE of colicin CE, the Pfu DNA polymerase has higher processivity and yield.

[0008] The invention introduces a heat-resistant dCE having DNA binding activity but not having DNA cutting activity into Pfu DNA polymerase, thereby significantly improving the amplification speed and amplification yield of Pfu DNA polymerase. The mutant dCE is transformed from the C-terminal DNA enzyme domain (CE2, CE7, CE8, CE9) of colicin and is named dCE2, dCE7, dCE8, and dCE9, respectively. The research of the invention shows that the four dCEs all have the function of improving the extension speed and amplification yield of Pfu DNA polymerase.

[0009] The second aspect of the present invention provides a highly processive Pfu DNA polymerase, specifically: a mutant dCE of colicin CE is fused to the N-terminus of the wild-type Pfu DNA polymerase protein sequence, wherein dCE is any of the following:

[0010] dCE2, the gene sequence of which is shown in SEQ ID NO.2;

[0011] dCE7, the gene sequence of which is shown in SEQ ID NO.3;

[0012] dCE8, the gene sequence of which is shown in SEQ ID NO.4;

[0013] dCE9, whose gene sequence is shown in SEQ ID NO.5.

[0014] The present invention names the Pfu DNA polymerase with a dCE label as dCE-Pfu.

[0015] Preferably, in the above-mentioned highly processive Pfu DNA polymerase, the wild-type Pfu DNA polymerase protein sequence is derived from the extreme thermophilic bacterium Pyrococcusfuriosus, and its gene sequence is shown in SEQ ID NO.1.

[0016] In one embodiment of the present invention, a mutant dCE7 was fused and expressed at the N-terminus of the Pfu DNA polymerase protein sequence from the extremely thermophilic bacterium Pyrococcusfuriosus to obtain a DNA polymerase dCE7-Pfu having an amino acid sequence as shown in SEQ ID NO. 6. The dCE7-Pfu amino acid sequence provided in the embodiment of the present invention carries a purification tag (His tag). In the specific implementation process, those skilled in the art can replace or modify the tag sequence according to actual needs to obtain a fusion protein with the same function as the dCE7-Pfu of the present invention, which still belongs to the protection scope of the present invention.

[0017] The third aspect of the present invention provides biological materials related to the highly processive Pfu DNA polymerase of the present invention, including but not limited to the following materials:

[0018] A) a gene encoding the highly processive Pfu DNA polymerase of the present invention;

[0019] b) a recombinant expression vector containing the gene described in a);

[0020] c) a recombinant cell containing the gene described in a) or the recombinant expression vector described in b) .

[0021] A fourth aspect of the present invention provides a method for preparing the highly processive Pfu DNA polymerase of the present invention, comprising the following steps:

[0022] S1, synthesizing the gene sequences of wild-type Pfu DNA polymerase and mutant dCE, and inserting them into pET23a vector step by step through T5-mediated DNA cloning method to obtain the recombinant expression vector pET23a-dCE-Pfu;

[0023] S2. The recombinant expression vector pET23a-dCE-Pfu was transformed into Escherichia coli to induce expression to obtain the target protein.

[0024] Preferably, the dCE-Pfu DNA polymerase gene coding sequence in step S1 is as shown in SEQ ID NO.1, and the gene sequence of the dCE protein is as shown in any one of SEQ ID NOs.1-4.

[0025] The fifth aspect of the present invention provides the use of the dCE-Pfu DNA polymerase of the present invention in DNA replication, specifically: constructing a reaction system containing dCE-Pfu DNA polymerase, a DNA template and primers, and achieving DNA replication through RCR.

[0026] The experimental data of the present invention show that the amplification speed and amplification yield of dCE-Pfu DNA polymerase are significantly improved compared with the wild-type Pfu DNA polymerase. In addition, when the dCE-Pfu DNA polymerase is dCE7-Pfu, the optimal K + The concentration is 16-18mM.

[0027] The sixth aspect of the present invention provides use of the dCE-Pfu DNA polymerase of the present invention in preparing a PCR amplification reagent or a kit.

[0028] The above-mentioned PCR amplification reagent or kit may also include other reagents and / or consumables for PCR amplification.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] The present invention has carried out molecular biological transformation on the Pfu DNA polymerase in the nucleic acid amplification technology, and for the first time found that the progressivity of the Pfu DNA polymerase can be significantly improved by fusing and expressing the mutant dCE of colicin CE, providing a new solution to the problem of low extension rate of the Pfu DNA polymerase; experimental results show that the amplification speed of the modified polymerase can reach about 8kb / min, which is much higher than the wild-type Pfu DNA polymerase (about 600bp / min), and the amplification yield is also significantly improved. The present invention also provides a specific preparation method and application method of the modified polymerase, laying a foundation for the practical application of dCE-Pfu DNA polymerase. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an SDS-PAGE image of samples during the recombinant expression and purification of dCE7-Pfu prepared in Example 1 of the present invention.

[0032] Figure 2 This is a comparison chart of the agarose gel electrophoresis detection results of the amplification products of commercial Pfu and dCE7-Pfu in Example 2 of the present invention.

[0033] Figure 3is K in the dCE7-Pfu reaction system in Example 3 of the present invention + Graph showing the effect of concentration on amplification yield. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0035] 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 application belongs.

[0036] In order to solve the problem of low extension rate of Pfu DNA polymerase in the prior art, the present invention provides a new solution, specifically: dCE is fused and expressed at the N-terminus of the wild-type Pfu DNA polymerase protein sequence, wherein the gene sequence of dCE is specifically: dCE2 (as shown in SEQ ID NO.2), dCE7 (sequence as shown in SEQ ID NO.3), dCE8 (sequence as shown in SEQ ID NO.4) and dCE9 (sequence as shown in SEQ ID NO.5).

[0037] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0038] The Escherichia coli BL21 (DE3) used in the following examples was purchased from Novagen, the primers used were commissioned to be synthesized by China Sangon Biotechnology Co., Ltd., the plasmid pET23a-dCE containing the dCE gene was commissioned to be synthesized by China Jinkairui Biotechnology Co., Ltd., and the vector pET23a used was purchased from Novagen.

[0039] The following examples take mutant dCE7 as an example to demonstrate the preparation of fusion protein dCE-Pfu and the effect of fusion expression of mutant dCE on the performance of Pfu DNA polymerase.

[0040] Example 1 Preparation of dCE-Pfu DNA polymerase

[0041] The wild-type Pfu DNA polymerase in this example is derived from the extremely thermophilic bacterium Pyrococcusfuriosus, and its gene coding sequence is shown in SEQ ID NO: 1. Specifically, dCE7-Pfu is prepared by the following steps:

[0042] (1) Construction of dCE7-Pfu expression vector.

[0043] The Pfu DNA polymerase gene coding sequence obtained from the extreme thermophilic bacterium Pyrococcusfuriosus is shown in SEQ ID NO.1, and the gene sequence of dCE7 is shown in SEQ ID NO.6. The above sequences were synthesized by Sangon Biotechnology Co., Ltd. and inserted into the pET23a vector step by step through the T5-mediated DNA cloning method. The recombinant vector was named pET23a-dCE7-Pfu.

[0044] (2) Recombinant expression and purification of dCE7-Pfu in E. coli.

[0045] The expected size of dCE7-Pfu is about 100 kDa, and its amino acid sequence is shown in SEQ ID NO.3.

[0046] The expression vector pET23a-dCE7-Pfu was transformed into Escherichia coli BL21 (DE3), and the transformants were selected and inoculated into LB liquid medium containing 50 mg / mL ampicillin sodium. The culture was shaken at 37°C and 220 rpm. When the OD of the culture medium reached 600 When the cell density reaches 0.6-0.8, add IPTG with a final concentration of 1mM, and induce the culture at 18℃ and 220rpm for 16-18h. Then, centrifuge at 6000rpm for 10min to collect the bacteria, wash the culture solution with Buffer A (20mM Tris-HCl pH8.2, 100mM KCl, 10mM imidazole, 0.1mMEDTA), and resuspend the bacteria in Buffer A (add PMSF with a final concentration of 1mM). Repeat the disruption with a high-pressure cell disruptor for 5 times, then centrifuge at 18000rpm for 10min, collect the supernatant, add DNase and RNase, incubate at 37℃ for 1h to remove DNA and RNA. Then, centrifuge the sample at 18000rpm for 20min, and transfer the supernatant to the pretreated gravity column.

[0047] The pretreatment method is as follows: 4 mL of nickel beads are drawn into a 60 mL protein purification gravity column, and the nickel beads are balanced with 3 volumes of sterilization buffer. After the supernatant is transferred to the gravity column, the sealing film is sealed, and it is incubated at 4 ° C for 1 h on a silent mixer, and then washed with 20 mM imidazole for 10 column volumes (added 3 times), followed by 50 mM, 100 mM, and 300 mM for 3 column volumes, and the effluent is collected for SDS-PAGE detection. The eluted fraction containing the high-purity target protein is ultrafiltered and concentrated using a 50 kDa ultrafiltration tube, and stored in Buffer B (20 mM Tris-HCl pH 8.2, 100 mM potassium glutamate, 0.1 mM EDTA, 1 mM DTT, 0.1% (v / v) Nonidet P40, 0.1% (v / v) Tween 20), and then aliquoted and quickly frozen in liquid nitrogen and stored at -80 ° C.

[0048] The SDS-PAGE results of the dCE7-Pfu purification process are shown in Figure 1 .

[0049] Example 2 dCE-Pfu extension rate detection

[0050] Using pRGEB32 as a template, commercial Pfu (Thermofisher) and dCE7-Pfu (prepared in Example 1) were used to amplify 2kb, 4kb, 6kb, 8kb and 10kb fragments, respectively. The primers used are shown in Table 1, the reaction system is shown in Table 2, and the PCR program is as follows: pre-denaturation at 97°C for 20s; denaturation at 97°C for 5s, extension at 72°C for 2min, 21 cycles; finally extension at 72°C for 7min, storage at 4°C

[0051] Table 1

[0052]

[0053] Table 2

[0054]

[0055]

[0056] The specific components of the Buffer in Table 2 are: 200 mM Tris-HCl (pH 8.8), 100 mM KCl, 100 mM (NH4)2SO4, 22 mM MgSO4, 1% (v / v) Triton X-100, and 1 mg / mL BSA.

[0057] After the reaction is completed, the PCR product is detected by agarose gel electrophoresis. Figure 2The results showed that the amplification rate of dCE7-Pfu reached about 8kb / min, which was much higher than that of Pfu DNA polymerase, and the amplification yield was also significantly improved.

[0058] Example 3d K of CE-Pfu reaction system + Concentration optimization

[0059] In this case, the rice genome was used as a template to optimize the reaction system of dCE-Pfu. The reaction system is shown in Table 1, and the PCR program is: 98℃ pre-denaturation for 2min; 98℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 20s, 26 cycles; finally 72℃ extension for 5min, 4℃ storage.

[0060] Table 3

[0061]

[0062] The difference between Buffer and Example 2 is that K + The concentration ranged from 8 mM to 20 mM, with each gradient increasing by 2 mM (two replicates for each gradient).

[0063] The results are as follows Figure 3 As shown, dCE7-Pfu is most suitable for the reaction K + The concentration is 16-18mM.

[0064] In summary, the present invention effectively improves the amplification rate and amplification yield of the modified DNA polymerase by fusing and expressing dCE at the N-terminus of the Pfu DNA polymerase protein sequence, and provides a new solution to the problem of low amplification rate of Pfu DNA polymerase.

[0065] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. An application of a highly processive Pfu DNA polymerase in DNA replication, characterized in that, The amino acid sequence of the highly processive Pfu DNA polymerase is shown in SEQ ID NO.6, which is obtained by fusing the mutant dCE7 of colicin CE to the N-terminus of the wild-type Pfu DNA polymerase protein sequence, and the wild-type Pfu DNA polymerase is derived from the extremely thermophilic bacterium Pyrococcusfuriosus; Specifically, the preparation method of the highly processive Pfu DNA polymerase comprises the following steps: S1, synthesizing the gene sequences of wild-type Pfu DNA polymerase and mutant dCE, and inserting them into pET23a vector step by step through T5-mediated DNA cloning method to obtain the recombinant expression vector pET23a-dCE-Pfu; S2, transforming the recombinant expression vector pET23a-dCE-Pfu into Escherichia coli, inducing expression to obtain the target protein; The application method is: constructing a reaction system containing dCE-Pfu DNA polymerase, DNA template and primers to perform RCR, wherein K + The concentration is 16-18mM.

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