A mutant Taq DNA polymerase and its applications

Abstract

A mutant Taq DNA polymerase and its application belong to the field of biotechnology. On the one hand, the present invention provides a mutant Taq DNA polymerase. Compared with the amino acid sequence of the wild-type Taq DNA polymerase, the mutant Taq DNA polymerase includes the mutation site D785 and / or the mutation site R573. On the other hand, the present invention provides the application of the mutant Taq DNA polymerase in the preparation of PCR reaction reagents. The Taq DNA polymerase mutant described in the present invention is a mutant obtained by predicting the activity of key mutation sites by AlphaFold3 and then verifying the expression. The double mutant strain obtained in the present invention provides a better Taq enzyme selection for genomic amplification.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a mutant Taq DNA polymerase and its application. Background Art

[0002] Polymerase chain reaction (PCR) is the most popular application in contemporary molecular biology and diagnostics. The key component of PCR is thermostable DNA polymerases, which synthesize new DNA complementary to the DNA template. Taq DNA polymerase is the most popular enzyme in most PCR applications due to its stability and low cost.

[0003] The wild-type Taq DNA polymerase is 832 amino acids in length and can be divided into two active domains: the 5'-3' exonuclease active domain is located at positions 1-291. The deletion of this domain does not cause Taq to lose its polymerase activity, but it has some effects on the catalytic performance of the enzyme; the polymerase active domain is located at positions 410-832. This domain resembles a right hand and includes the Thumb, Palm, and Fingers structures. The Palm domain contains the most important catalytic sites Asp610, Asp785, and Glu786, which is the core region of polymerase activity. The Fingers domain and Thumb domain play a key role in substrate specificity and maintaining the spatial structure between Taq enzyme and substrate. The domain formed by the amino acids at positions 292-423 between the two active regions resembles the 3'-5' exonuclease domain of pol I, but there is no sequence homology between them, so Taq enzyme does not have 3'-5' exonuclease activity.

[0004] With the development of biotechnology, the industry's requirements for Taq DNA polymerase are also constantly increasing. More and more Taq DNA polymerase mutants with different characteristics have been screened out. The R660D / F667Y double mutation can significantly improve the incorporation rate of the ddNTP incorporation activity group of the enzyme, eliminate the preference for ddGTP incorporation, and greatly improve the quality and accuracy of Sanger sequencing. The SYBR Green I dye required for qPCR is an inhibitor that inhibits the activity of Taq enzyme; in the field of medical diagnosis, there are also related inhibitors in whole blood samples. By introducing E189K, E230K, E708Q, and E507K, the tolerance of Taq DNA polymerase to inhibitors such as SYBR Green I, whole blood, and SDS can be greatly improved. At the same time, introducing E189K, E230K, and E508K can greatly improve the amplification rate (the highest amplification rate can reach 25 s / kb), introducing H28R can increase the amplification length, and introducing D452N and G504S is beneficial for Taq enzyme to resist the USP hairpin structure in the template. These mutants have greatly enriched the application scope of Taq.

[0005] Directed evolution of enzyme proteins is the most important method for screening mutants. However, this method requires the design of an effective high-throughput screening method to successfully obtain excellent mutants. At the same time, the experimental process requires the construction of a mutant library and the screening of a large number of mutant clones. The whole experimental process is relatively complex, requiring a large amount of labor and time costs, and the mutation results are not predictable. With the rapid development of AI technology, the emergence of AlphaFold3 provides a powerful tool for our mutant screening. AlphaFold3 uses deep learning algorithms to train models by analyzing a large amount of protein structure data, can accurately predict the protein structure from the amino acid sequence, analyze the binding activity between the protein and the substrate, and can quickly and efficiently complete the mutant screening, which is the future development trend. Summary of the Invention

[0006] Aiming at the existing problems, the purpose of the present invention is to design and provide a technical solution for a mutant Taq DNA polymerase and its application.

[0007] The present invention first analyzes the binding sites between the wild-type Taq enzyme and the substrate (template-prime complex) (such as Figure 1 ), and finds that the sites where Taq enzyme interacts with DNA include: N485, S515, A516, R536, E537, T539, K540, S543, A568, R573, S575, S577, N580, N583, V586, E615, F667, Y671, R746, N750, D785. Subsequently, the complex structure of Taq enzyme with double-stranded DNA and single-base substrate (including analogs) (PDB: 5E41) is further analyzed. This structure (such as Figure 2 ) shows the binding sites of Taq with different incorporated substrates, including R587, D610, Y611, R660, K663, T664, D785. The complex structure of Taq enzyme with dTMeTP (PDB: 3M8R) is continuously analyzed, and the binding sites (such as Figure 3 ) are: R573, I614, E615, Q754, A757, H784, D785. Finally, the complex structure of Taq enzyme with ddTTP (PDB: 3RRH) is analyzed, and the binding sites (such as Figure 4It includes: R587, Y671, F667. Finally, five key binding sites, namely R573, R587, Y671, A757, and D785, were screened out. Through the analysis of mutants in the prior art, it was found that the common mutation methods in Taq enzyme mutants include R mutated to K / D, Y mutated to F / N, A mutated to L / E, D mutated to G / K, etc. The present invention selected ten mutation methods: R573K, D785G, A757L, R573D, R587D, Y671F / N, R587K, A757E, D785K. The binding ability between these ten mutants and the substrate was predicted and analyzed by AlphaFold3. The present invention found that the mutations of R573K, D785G, and A757L would increase the binding ability of Taq enzyme to DNA; the mutations of R573D, R587D, and Y671F / N would decrease the binding ability of Taq enzyme to DNA; the mutations of R587K, A757E, and D785K had no obvious change.

[0008] Furthermore, the present invention performed protein expression on the ten mutant strains obtained by the above screening, and seven mutants were successfully expressed. After verifying the enzyme activity of Taq enzyme, the present invention screened out two significant mutation sites, D785G and R573K. Through the expression of mutants and the verification of enzyme activity, the present invention found that the enzyme activity of the D785G mutant strain was increased by 3 times compared with the wild type, and the enzyme activity of the R573K mutant strain was increased by 1.8 times. At the same time, the present invention constructed a double mutant strain of D785G / R573K and found that the enzyme activity of its double mutant strain was slightly lower than that of the D785G mutant strain, but it was found in subsequent verification experiments that it had a significant improvement in the amplification length and the amplification of high GC content genomes.

[0009] The present application specifically adopts the following technical solutions to achieve:

[0010] In the first aspect of the present invention, a mutant Taq DNA polymerase is provided. Compared with the amino acid sequence of the wild-type Taq DNA polymerase, the mutant Taq DNA polymerase includes the mutation site D785 and / or the mutation site R573.

[0011] Furthermore, the amino acid sequence of the mutant Taq DNA polymerase has more than 90% homology with the amino acid sequence of the wild-type Taq DNA polymerase.

[0012] Preferably, the amino acid sequence of the mutant Taq DNA polymerase has more than 95% homology with the amino acid sequence of the wild-type Taq DNA polymerase.

[0013] More preferably, the amino acid sequence of the mutant Taq DNA polymerase has more than 99% homology with the amino acid sequence of the wild-type Taq DNA polymerase.

[0014] Furthermore, compared with the amino acid sequence of wild-type Taq DNA polymerase, the mutant Taq DNA polymerase includes the mutation site D785G and / or the mutation site R573K.

[0015] The second aspect of the present invention provides a gene encoding the mutant Taq DNA polymerase described above.

[0016] The third aspect of the present invention provides a recombinant expression vector containing the above gene.

[0017] The fourth aspect of the present invention provides a recombinant bacterium containing the above recombinant expression vector.

[0018] The fifth aspect of the present invention provides an application of the above mutant Taq DNA polymerase in the preparation of PCR reaction reagents.

[0019] Advantages of the present invention:

[0020] The mutant described in the present invention is obtained by simulating the binding of Taq enzyme and substrate, analyzing the core binding site, and then simulating the enzyme / substrate binding activity through AlphaFold3. After experimental verification, it is determined that the enzyme activities of two mutants with improved binding ability have been improved. Surprisingly, the combined mutation of the two sites does not significantly improve the enzyme activity, but the amplification length and the amplification ability of high-GC-content templates have been improved. This may be related to the distribution of the two sites. The D785 site is the core binding site located in the Palm region, and the mutation of this site is more conducive to the improvement of enzyme activity. The R573 site is in the finger region, which is more to stabilize the enzyme / substrate complex structure and is more conducive to the amplification of GC-content templates. The double mutant strain obtained in the present invention provides a better Taq enzyme choice for genomic amplification. Description of the drawings

[0021] Figure 1 Analysis of the binding site between wild-type Taq enzyme and substrate;

[0022] Figure 2 Structures of Taq enzyme complex with double-stranded DNA and single-base substrates. In the figure, A) is the complex structure of Taq enzyme and dUTP analog, B) is the complex structure of Taq enzyme and dCTP, C) is the complex structure of Taq enzyme and dATP analog, D) is a partial enlarged view of the core region of the binding site, E) is a partial enlarged view of the core region of the binding site, F) is a partial enlarged view of the core region of the binding site;

[0023] Figure 3 Binding site of Taq enzyme and dTMeTP complex structure;

[0024] Figure 4Binding site of the Taq enzyme-ddTTP complex structure (PDB: 3RRH);

[0025] Figure 5 PCR screening results of 7 single mutant Taq enzymes;

[0026] Figure 6 PCR screening results of double mutant strains and their corresponding single mutant Taq enzymes;

[0027] Figure 7 Results of amplifying high GC content templates of different lengths by double mutant strains. Detailed implementation mode

[0028] The present invention will be further described below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Any simple improvement to the preparation method of the present invention under the premise of the concept of the present invention belongs to the protection scope of the present invention. The experimental methods without specific conditions described in the following examples are usually carried out according to the well-known means in the art. The test materials used in the following examples are all obtained from conventional biochemical reagent stores without special instructions.

[0029] In the present invention, each substitution is represented by a triplet: letter - number - letter, where the number represents the position of the mutant amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number represents the amino acid used to replace the amino acid before the number.

[0030] The wild-type Taq DNA polymerase in the present invention is an existing enzyme, and its amino acid sequence is shown in SEQ ID NO.1.

[0031] Example 1: Ten single point mutations cloning

[0032] According to the method of introducing site-directed mutations in conventional molecular cloning, mutant primer pairs were designed, and the wild-type Taq enzyme expression plasmid was amplified with the mutant primer pairs. After digesting the template plasmid with DpnI, it was transformed into BL21 DE3 competent cells, plated and cultured overnight. After picking clones and sequencing, mutant clones could be obtained.

[0033] The mutation site of Mut1 is: R573K

[0034] The mutation site of Mut2 is: R573D

[0035] The mutation site of Mut3 is: R587D

[0036] The mutation site of Mut4 is: R587K

[0037] The mutation site of Mut5 is: D785K

[0038] The mutation site of Mut6 is: D785G

[0039] The mutation site of Mut7 is: Y671N

[0040] The mutation site of Mut8 is: Y671F

[0041] The mutation site of Mut9 is: A757E

[0042] The mutation site of Mut10 is: A757L.

[0043] Table 1 Primer sequences of mut1 - mut10 mutants

[0044]

[0045] The primers used for mutant construction are shown in Table 1, and the serial numbers of the mutant names and primer names are the same. For example, the primers required for the Mut1 mutant are Mut1 - F / Mut1 - R. The system and procedure of each group of PCR are shown in Table 2:

[0046] Table 2 PCR system and amplification procedure

[0047]

[0048] After PCR, add 5.6 μL of rCutsmart buffer and 1 μL of DpnI fast digestion enzyme, and incubate at 37 °C for 30 min. The reaction product after DpnI digestion is purified and recovered using the SanPrep column PCR product purification kit (B518141); take 25 ng of the purified plasmid and transform it into BL21 DE3 competent cells; after correct sequencing, the Taq enzyme mutant expression strain can be obtained. The amino acid sequences of Mut1 - 10 are shown in SEQ ID NO.2 - 11. Finally, Mut1, Mut2, Mut4, Mut5, Mut6, Mut9, and Mut10 were successfully expressed and purified. The PCR screening results of the 7 single - mutant Taq enzymes, and the system and procedure of each group of PCR are shown in Table 3:

[0049] Table 3 PCR system and amplification procedure

[0050]

[0051] Electrophoresis separation on 1% agarose gel is as Figure 5 shown. From the gel picture bands, we can qualitatively judge that the enzyme activities of Mut1 and Mut6 are significantly improved compared with the wild - type. Subsequently, the specific enzyme activities of all mutants were detected using the [3H] - dTTP isotope method.

[0052] Example 2: Enzyme Activity Detection of Seven Mutant Taq Enzymes and Wild-Type Taq Enzyme

[0053] The enzyme activities of the 7 Taq enzyme mutants and the wild-type obtained in Example 1 were detected by the [3H]-dTTP isotope method, and the results are shown in Table 4 below.

[0054] Table 4 Enzyme Activity Data of Seven Mutants

[0055]

[0056] Example 3: Construction and Enzyme Activity Determination of Double Mutants

[0057] The double mutants were constructed according to the method of Example 1, using Mut1 as the sequence template and the Mut6-F / Mut6-R primer pair for construction. After correct sequencing, induction expression and purification were carried out. The amino acid sequence of Mut11 is shown in SEQ ID NO.12.

[0058] The mutation sites of Mut11 are: D785G / R573K;

[0059] The PCR screening method for Mut11 double mutants was carried out according to Table 3 in Example 1, and the separation results were obtained by running on a 1% agarose gel as Figure 6 shown. From the PCR amplification bands, the enzyme activity of the double mutants did not increase and was slightly lower than that of Mut6.

[0060] Similarly, the enzyme activity of the double mutants was determined by the [3H]-dTTP isotope detection method, and the results are shown in Table 4. The enzyme activity data was consistent with the agarose gel electrophoresis results. The enzyme activity of the Mut11 double mutant strain was higher than that of Mut1 but slightly lower than that of Mut6. This may be because D785G is at the core site of the catalytic site, and the increase in enzyme activity is mainly caused by this site.

[0061] Table 5 Enzyme Activity Data of Mut11 Mutants

[0062]

[0063] Example 4: Amplification of Mut11 Mutant with High GC Content and Amplification of Different Lengths

[0064] The PCR procedure and system for this implementation scheme are shown in Table 6, and the required primers are shown in Table 7. In this example, the templates for amplification are plasmid - 872, plasmid - 2101, and plasmid 5073. The corresponding sequences in NCBI were synthesized by GenScript and cloned into the PUC57 plasmid. The cloning restriction enzyme sites are NcoI and XhoI. Among them, the 70% GC nucleic acid sequence of sequence 872 is shown as SEQ ID NO.13; the 60% GC nucleic acid sequence of sequence 2101 is shown as SEQ ID NO.14; the 60% GC nucleic acid sequence of sequence 5073 is shown as SEQ ID NO.15.

[0065] Table 6 Amplification System and Procedure for Sequences of Different Lengths and High GC Content

[0066]

[0067] Table 7 PCR Amplification Primers

[0068]

[0069] Run nucleic acid electrophoresis on 1% agarose gel, and the result is as Figure 7 shown: The Mut11 double mutant strain performs better in the amplification of 872bp and 5073bp templates with high GC content. The wild - type Taq enzyme cannot effectively amplify the 5073bp template with high GC content, indicating that Mut11 can amplify longer fragments in templates with high GC content. The above results show that the R573K mutation is more beneficial for Taq enzyme to amplify templates with high GC content, especially those with more consecutive GCs. This enzyme can provide a better choice for the amplification of some plant genomic sequences.

Claims

1. A mutant Taq DNA polymerase, characterized in that Compared with the amino acid sequence of the wild-type Taq DNA polymerase, the mutant Taq DNA polymerase has mutation sites D785G and R573K. The amino acid sequence of the mutant Taq DNA polymerase is shown in SEQ ID NO.

12.

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

3. A recombinant expression vector comprising the gene according to claim 2. A recombinant bacterium comprising the recombinant expression vector according to claim 3.

5. Use of the mutant Taq DNA polymerase according to claim 1 in preparing PCR reaction reagents.

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