A terminal deoxynucleotidyl transferase mutant with high thermal stability and its application

Abstract

The present invention belongs to the technical field of enzyme engineering, and particularly relates to a terminal deoxynucleotidyl transferase mutant with high thermal stability and its application. The terminal deoxynucleotidyl transferase mutant is obtained by multiple point mutations on the amino acid sequence shown in SEQ ID NO.1. After structurally truncating the wild-type terminal deoxynucleotidyl transferase TdT derived from Zonotrichia albicollis, and then performing multiple point mutations, the present invention obtains a series of mutants with improved heat resistance. These mutants have better thermal stability and higher unfolding temperatures, and can play an important role in DNA enzymatic synthesis.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a terminal deoxynucleotidyl transferase mutant with high thermal stability and its application. Background Art

[0002] DNA synthesis is a key technology in the fields of life science, biomedicine, materials science, and information storage. Currently, DNA synthesis mainly relies on chemical synthesis methods, and there are insurmountable limits in terms of synthesis length and cost, which cannot meet the growing demands in the field of biomanufacturing. Enzymatic DNA synthesis, on the other hand, has unparalleled potential in terms of synthesis speed, length, efficiency, and cost compared to chemical synthesis, and has become the forefront direction for the development of DNA synthesis technology.

[0003] Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase that can randomly add deoxynucleotides to a primer without relying on a template. Based on the template-independent property of terminal deoxynucleotidyl transferase, it can be used for DNA synthesis. When the growing single-stranded DNA molecule reaches a certain length, a secondary structure will form. However, TdT has poor activity towards substrates with a secondary structure at the 3′-OH terminus, resulting in reduced extension efficiency. High temperature can disrupt the hairpin structure of the primer strand and restore it to a single strand that is easy to extend. However, TdT has poor stability, and high temperature will cause the loss of enzyme activity and the loss of catalytic reaction function. Therefore, improving the thermal stability of TdT is of great significance in DNA enzymatic synthesis. Summary of the Invention

[0004] Aiming at the defect of poor heat resistance of terminal deoxynucleotidyl transferase in the prior art, the present invention provides a terminal deoxynucleotidyl transferase mutant with high thermal stability and its application. The specific technical solutions are as follows:

[0005] In the first aspect, the present invention provides a terminal deoxynucleotidyl transferase mutant with high thermal stability, which is one of the following mutant forms:

[0006] (1) Mutated from the amino acid sequence shown in SEQ ID NO.2 by S116A / P301A / L50V / F40R / C173W / A257C / V70F / N192E / L166Q / A322R / Q104L / Y17H / S57T / N91K / I39G / E84H / R89E / K35N / K11R / I82L / G346T / E43A / Y302F / V27I / V164I / S7A / R338T / I232Q / V360I / R340K / N170E / L45R / K339Q / K100Q / I197V / E79Q, and the amino acid sequence is as shown in SEQ ID NO.3;

[0007] (2) It is obtained by mutating the amino acid sequence shown in SEQ ID NO.2 with S116A / P301A / L50V / F40R / C173W / A257C / V70F / N192E / L166Q / A322R / Q104L / Y17H / S57T / N91K / I39G / E84H / R89E / K35N / K11R / I82L / G346T / E43A / Y302F / V27I / V164I / S7A / R338T / I232Q / V360I / R340K / N170E / L45R / K339Q / K100Q / I197V / E79Q / V237D / S139T / Q73H / Q240K / L147Q / K20R / E239S / D358E, and the amino acid sequence is as shown in SEQ ID NO.4;

[0008] (3) It is obtained by mutating the amino acid sequence shown in SEQ ID NO.3 with G314N / Q232I / I328L / E275K / I183M / D76R;

[0009] (4) It is obtained by mutating the amino acid sequence shown in SEQ ID NO.3 with G314N / Q232I / I328L / E275K / I183M / D76R / T270N / R311K / M28L / E92R / A101T;

[0010] (5) It is obtained by mutating the amino acid sequence shown in SEQ ID NO.3 with G314N / Q232I / I328L / E275K / I183M / D76R / T270N / R311K / M28L / E92R / A101T / V237E / T174A / Q240K / K20R / F297W / D358E / C271Y;

[0011] (6) It is obtained by mutating the amino acid sequence shown in SEQ ID NO.4 with G314N / E361P / Q232I;

[0012] (7) It is obtained by mutating the amino acid sequence shown in SEQ ID NO.4 with G314N / E361P / Q232I / I183M;

[0013] (8) It is obtained by mutating the amino acid sequence shown in SEQ ID NO.4 with G314N / E361P / Q232I / I183M / I328L;

[0014] (9) It is obtained by mutating the amino acid sequence shown in SEQ ID NO.4 with G314N / E361P / Q232I / I183M / I328L / N31Y;

[0015] (10) obtained by subjecting the amino acid sequence shown in SEQ ID NO. 4 to G314N / E361P / Q232I / I183M / I328L / N31Y / E275K / D76R mutations;

[0016] (11) The amino acid sequence shown in SEQ ID NO.4 was mutated to G314N / E361P / Q232I / I183M / I328L / N31Y / E275K / D76R / R311K / I288V / C271Y / T174A / K91E / F297Y / E92R / E278D / I294V, and the amino acid sequence is shown in SEQ ID NO.5.

[0017] In the present invention, the terminal deoxynucleotidyl transferase TdT derived from Zonotrichia albicollis is used, and Alphafold 2 is used to construct the three-dimensional structure of TdT. Based on the model scoring, a suitable structure is selected, and the Loop part in its protein structure is truncated to obtain a truncated terminal deoxynucleotidyl transferase. On the basis of the truncated terminal deoxynucleotidyl transferase, the pross online tool based on the energy function is used to predict the mutation site, and multiple point mutations are performed to obtain a terminal deoxynucleotidyl transferase mutant that is more resistant to high temperatures, and its unfolding temperature is increased by up to 9.1°C.

[0018] In a second aspect, the present invention also provides a terminal deoxynucleotidyl transferase mutant obtained by subjecting the above mutant to R189L / K191G mutation.

[0019] Furthermore, the mutant with the amino acid sequence as shown in SEQ ID NO.5 is subjected to R189L / K191G mutation to obtain the mutant.

[0020] Studies have shown (Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase|ACS Catalysis) that the R335L / K337G combination mutation of the wild-type terminal deoxynucleotidyl transferase can increase the activity of 3′-ONH2-dNTP. 3′-ONH2 can block the continuous addition of nucleotides, and then remove 3′-ONH2 by NaNO2 to unblock it, which can achieve controlled addition of nucleotides. In the present invention, since the N-terminal Loop region of the wild-type terminal deoxynucleotidyl transferase is truncated, the original R335L / K337G combination mutation is R189L / K191G in the present invention.

[0021] In a third aspect, the present invention provides a gene encoding the terminal deoxynucleotidyl transferase mutant described in the first and second aspects.

[0022] In a fourth aspect, the present invention provides a recombinant vector comprising the above-mentioned encoding gene.

[0023] In a fifth aspect, the present invention provides a genetically engineered bacterium comprising the above-mentioned encoding gene or comprising the above-mentioned recombinant vector.

[0024] Further, the genetically engineered bacterium is Escherichia coli.

[0025] Further, the genetically engineered bacterium is E. coli BL21(DE3).

[0026] In a sixth aspect, the present invention provides the application of the terminal deoxynucleotidyl transferase mutant in the first and second aspects in enzymatic DNA synthesis.

[0027] In a seventh aspect, the present invention provides a method for enzymatically synthesizing DNA, which uses deoxyribonucleotides as substrates and synthesizes DNA under the catalytic action of the terminal deoxynucleotidyl transferase mutant described in the second aspect.

[0028] Further, the 3'-end of the deoxyribonucleotide is modified with a reversible blocking group; the terminal deoxynucleotidyl transferase mutant is mutated to improve the activity of binding to deoxyribonucleotides modified with a reversible blocking group at the 3'-end.

[0029] The terminal deoxynucleotidyl transferase mutant with high thermal stability provided in the first aspect of the present invention, after undergoing the process of truncating the Loop and multiple-point mutation, has the characteristic of high temperature resistance, and can be mutated to adapt to substrates, so as to synthesize DNA using deoxyribonucleotides with different types of reversible blocking groups such as O-amino, O-allyl, O-azide, O-phosphate group, etc.

[0030] In an eighth aspect, the present invention provides a method for enzymatically synthesizing DNA, using deoxyribonucleotides modified with O-amino groups at the 3'-end as substrates.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] By structurally truncating the wild-type terminal deoxynucleotidyl transferase TdT derived from Zonotrichia albicollis and then performing multiple-point mutation, the present invention obtains a series of mutants with improved heat resistance. These mutants have better thermal stability and higher unfolding temperature, and can play an important role in DNA enzymatic synthesis. Brief Description of the Drawings

[0033] Figure 1 Schematic diagram of the protein structure of TdT; among them, the circled part is the Loop structure of the TdT protein.

[0034] Figure 2 Gel electrophoresis patterns of different mutants; among them, WT-S represents truncated terminal deoxynucleotidyl transferase, N represents the length of the primer, N+1 represents the length of the primer extended by one base, M1-M7 represent different terminal deoxynucleotidyl transferase mutants, and 37, 56, 58, 60 represent the incubation temperatures of the protein.

[0035] Figure 3 Gel electrophoresis patterns of different mutants; among them, N represents the length of the primer, N+1 represents the length of the primer extended by one base, M1-1-M2-9 represent different terminal deoxynucleotidyl transferase mutants, and 37, 62, 64, 66 represent the incubation temperatures of the protein.

[0036] Figure 4 Gel electrophoresis patterns of different mutants; among them, N+1 represents the length of the primer extended by one base, WT and LG represent the mutant WT-S / LG, M2-8 and LG represent the mutant M2-8 / LG, and 37°C and 58°C represent the incubation temperatures of the protein.

[0037] Figure 5 Extension effects of M2-8 / LG and WT-S / LG, among which, BIO-52 is the name of the starting primer, and G, T, C, A represent 3ˊ-ONH2-dGTP, 3ˊ-ONH2-dTTP, 3ˊ-ONH2-dCTP, 3ˊ-ONH2-dATP respectively. Detailed implementation manners

[0038] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and only a part of the embodiments of the present invention, rather than all the embodiments.

[0039] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the scope of protection of the present invention.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in this field and can be obtained through commercial channels. The experimental methods without specifying detailed conditions are carried out according to conventional experimental methods or according to the operation manuals recommended by the suppliers.

[0041] In the following examples, the original terminal deoxynucleotidyl transferase (TdT) used was derived from Zonotrichia albicollis, and its amino acid sequence is shown in SEQ ID NO.1; the truncated terminal deoxynucleotidyl transferase obtained based on the original terminal deoxynucleotidyl transferase has an amino acid sequence shown in SEQ ID NO.2; the amino acid sequence of mutant M1 is shown in SEQ ID NO.3; the amino acid sequence of mutant M2 is shown in SEQ ID NO.4; the amino acid sequence of mutant M2-8 is shown in SEQ ID NO.5.

[0042] SEQ ID NO.1:

[0043] MDRFKAPAVISQRKRQKGLHSPKLSCSYEIKFSNFVIFIMQRKMGLTRRMFLMELGRRKGFRVESELSDSVTHIVAENNSYLEVLDWLKGQAVGDSSRFELLDISWFTACMEAGRPVDSEVKYRLMEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0044] SEQ ID NO.2:

[0045] MKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVE PWERNA

[0046] SEQ ID NO.3:

[0047] MKVSQYACQRRTTLNNHNKKFTDAFEIMAENYEFNENEGRCLAFRRAASVLKSLPFTVTRMKDIQGLPCFGDQVRDIIQEILEHGESSEVKEVLNDERYQAFKLFTSVFGVGVKTAEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAISQIVKETVWTFLPDALVTITGGFRRGKEIGHDVDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIQESTFVKEQLPSRKVDAMDHFQKCFCILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYAFALLGWTGSRQFGRDLRRYARHERKMILDNHGLYDRTQKIFLKATSEEEIFAHLGLDYIEPWERNA

[0048] SEQ ID NO.4:

[0049] MKVSQYACQRRTTLNNHNKRFTDAFEIMAENYEFNENEGRCLAFRRAASVLKSLPFTVTRMKDIQGLPCFGDHVRDIIQEILEHGESSEVKEVLNDERYQAFKLFTSVFGVGVKTAEKWYRMGLRTVEEVKADKTLKLTKMQKAGLQYYEDLVSCVSKAEADAISQIVKETVWTFLPDALVTITGGFRRGKEIGHDVDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIQESTFDKSKLPSRKVDAMDHFQKCFCILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYAFALLGWTGSRQFGRDLRRYARHERKMILDNHGLYDRTQKIFLKATSEEEIFAHLGLEYIEPWERNA

[0050] SEQ ID NO.5:

[0051] MKVSQYACQRRTTLNNHNKRFTDAFEIMAEYYEFNENEGRCLAFRRAASVLKSLPFTVTRMKDIQGLPCFGDHVRRIIQEILEHGESSEVERVLNDERYQAFKLFTSVFGVGVKTAEKWYRMGLRTVEEVKADKTLKLTKMQKAGLQYYEDLVSCVSKAEADAISQIVKETVWAFLPDALVTMTGGFRRGKEIGHDVDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFDKSKLPSRKVDAMDHFQKCFCILKLYQPRVDNSTYNTSKQLDMAEVKDWKAVRVDLVVTPYEQYAFALLGWTGSKQFNRDLRRYARHERKMLLDNHGLYDRTQKIFLKATSEEEIFAHLGLEYIPPWERNA。

[0052] Construction of Recombinant Bacteria with Truncated Terminal Deoxynucleotidyl Transferase in Example 1

[0053] The original terminal deoxynucleotidyl transferase (TdT, hereinafter also referred to as WT-L, the amino acid sequence is shown in SEQ ID NO.1) was synthesized by Tsingke Biotechnology Co., Ltd. and inserted between the restriction enzyme sites BamH I and HindIII of the vector pET28a to obtain the recombinant plasmid pET28a-TdT. The recombinant plasmid was transformed into E.coli BL21(DE3) competent cells by heat shock method. After correct sequencing verification, the recombinant strain E.coli BL21(DE3)-pET28a-TdT of the original terminal deoxynucleotidyl transferase was obtained.

[0054] The three-dimensional structure of TdT was constructed using Alphafold 2. Based on the model scoring, a suitable structure was selected, and the structure of TdT was observed with pymol. As Figure 1 shown, the first 147 amino acids at the N-terminus were designated as the Loop region, and the N-terminally truncated terminal deoxynucleotidyl transferase (hereinafter also referred to as TdT-WT-S) was constructed. Appropriate primers were designed for whole plasmid PCR.

[0055] Table 1 Primers used to construct the truncated terminal deoxynucleotidyl transferase

[0056]

[0057] The primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0058] PCR amplification system:

[0059]

[0060] PCR amplification conditions:

[0061] 1) Pre-denaturation: 98°C for 3 min;

[0062] 2) Denaturation: 98°C for 10 s; Annealing: 60°C for 15 s; Extension: 72°C for 1 min; A total of 30 cycles;

[0063] 3) Post-extension: 72°C for 5 min;

[0064] 4) Store at 4°C.

[0065] Transformation and verification:

[0066] The above PCR product was directly transformed into E.coli BL21(DE3) competent cells by heat shock method. After correct sequencing verification, the truncated terminal deoxynucleotidyl transferase strain E.coli BL21(DE3)-TdT-WT-S was obtained.

[0067] Example 2 Construction of mutants

[0068] The stability-improving mutants of the truncated terminal deoxynucleotidyl transferase TdT-WT-S obtained in Example 1 were predicted using the ProSS online tool based on the energy function, and the stability-improving mutants M1 to M7, M1-1 to M1-6, and M2-1 to M2-9 were obtained.

[0069] Mutant M1 was obtained by mutating the truncated terminal deoxynucleotidyl transferase obtained in Example 1 with S116A / P301A / L50V / F40R / C173W / A257C / V70F / N192E / L166Q / A322R / Q104L / Y17H / S57T / N91K / I39G / E84H / R89E / K35N / K11R / I82L / G346T / E43A / Y302F / V27I / V164I / S7A / R338T / I232Q / V360I / R340K / N170E / L45R / K339Q / K100Q / I197V / E79Q. Mutant M2 was obtained by mutating the truncated terminal deoxynucleotidyl transferase obtained in Example 1 with S116A / P301A / L50V / F40R / C173W / A257C / V70F / N192E / L166Q / A322R / Q104L / Y17H / S57T / N91K / I39G / E84H / R89E / K35N / K11R / I82L / G346T / E43A / Y302F / V27I / V164I / S7A / R338T / I232Q / V360I / R340K / N170E / L45R / K339Q / K100Q / I197V / E79Q / V237D / S139T / Q73H / Q240K / L147Q / K20R / E239S / D358E. Mutant M3 was obtained by mutating the truncated terminal deoxynucleotidyl transferase obtained in Example 1 with S116A / P301A / L50V / F40R / C173W / A257C. Mutant M4 was obtained by mutating the truncated terminal deoxynucleotidyl transferase obtained in Example 1 with S116A / P301A / L50V / F40R / C173W / A257C / V70I / N192E / L166Q / A322R. Mutant M5 was obtained by mutating the truncated terminal deoxynucleotidyl transferase obtained in Example 1 with S116A / P301A / L50V / F40R / C173W / A257C / V70I / N192E / L166Q / A322R / Q104L. Mutant M6 was obtained by mutating the truncated terminal deoxynucleotidyl transferase obtained in Example 1 with S116A / P301A / L50V / F40R / C173W / A257C / V70F / N192E / L166Q / A322R / Q104L / Y17H / S57T / N91K / I39G / E84H.Mutant M7 was obtained by mutating the truncated terminal deoxynucleotidyl transferase obtained in Example 1 with S116A / P301A / L50V / F40R / C173W / A257C / V70F / N192E / L166Q / A322R / Q104L / Y17H / S57T / N91E / I39G / E84H / R89E / K35N / K11R / I82L / G346T / E43A / Y302F / V27I / V164I / S7A / R338T / I232Q.

[0070] Mutant M1-1 was obtained by mutating Mutant M1 with G314N. Mutant M1-2 was obtained by mutating Mutant M1 with G314N / Q232I. Mutant M1-3 was obtained by mutating Mutant M1 with G314N / Q232I / I328L / E275K. Mutant M1-4 was obtained by mutating Mutant M1 with G314N / Q232I / I328L / E275K / I183M / D76R. Mutant M1-5 was obtained by mutating Mutant M1 with G314N / Q232I / I328L / E275K / I183M / D76R / T270N / R311K / M28L / E92R / A101T. Mutant M1-6 was obtained by mutating Mutant M1 with G314N / Q232I / I328L / E275K / I183M / D76R / T270N / R311K / M28L / E92R / A101T / V237E / T174A / Q240K / K20R / F297W / D358E / C271Y.

[0071] Mutant M2-1 was obtained by performing G314N / E361P mutation on mutant M2. Mutant M2-2 was obtained by performing G314N / E361P / Q232I mutation on mutant M2. Mutant M2-3 was obtained by performing G314N / E361P / Q232I / I183M mutation on mutant M2. Mutant M2-4 was obtained by performing G314N / E361P / Q232I / I183M / I328L mutation on mutant M2. Mutant M2-5 was obtained by performing G314N / E361P / Q232I / I183M / I328L / N31Y mutation on mutant M2. Mutant M2-6 was obtained by performing G314N / E361P / Q232I / I183M / I328L / N31Y / E275K / D76R mutation on mutant M2. Mutant M2-7 was obtained by performing G314N / E361P / Q232I / I183M / I328L / N31Y / E275K / D76R / R311K / I288V / C271Y / S4P mutation on mutant M2. Mutant M2-8 was obtained by performing G314N / E361P / Q232I / I183M / I328L / N31Y / E275K / D76R / R311K / I288V / C271Y / T174A / K91E / F297Y / E92R / E278D / I294V mutation on mutant M2. Mutant M2-9 was obtained by performing G314N / E361P / Q232I / I183M / I328L / N31Y / E275K / D76R / R311K / I288V / C271Y / T174A / K91E / F297W / E92R / E278D / V216I / V153S / T270N / S239T / S165A / Q276E / M28L / M249L / K344H / F56Y / D291H mutation on mutant M2.

[0072] Based on WT-S and M2-8, R189L / K191G mutation was performed to obtain WT-S / LG and M2-8 / LG.

[0073] The mutant genes were synthesized by Tsingke Biological Engineering Co., Ltd. and inserted between the restriction enzyme sites BamH I and Hind III of vector pET28a to obtain recombinant plasmids.

[0074] Example 3 Expression and Purification of Different Terminal Deoxynucleotidyl Transferase Mutants

[0075] The plasmid carrying the genes of different terminal deoxynucleotidyl transferase mutants (M1-7, M1-1-M1-6, M2-1-M2-9, WT-S / LG, M2-8 / LG) or the gene of truncated terminal deoxynucleotidyl transferase WT-S was transformed into E. coli BL21(DE3) competent cells by heat shock method. After correct sequencing verification, recombinant bacteria containing different terminal deoxynucleotidyl transferase mutant genes were obtained.

[0076] The obtained recombinant strains were spread on LB solid medium containing 50 μg / mL kanamycin (Kana) and cultured in a 37 °C constant temperature incubator for 12 h. Single colonies were picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL Kana and cultured at 37 °C and 220 rpm for 8-12 h. Then, they were transferred to 50 mL fresh LB liquid medium containing 50 μg / mL Kana at an inoculation amount of 2% and cultured with shaking at 37 °C and 220 rpm until the OD 600 reached about 0.8, and IPTG with a final concentration of 1 mM was added, followed by induction culture at 18 °C and 220 rpm for 16 h.

[0077] After the culture was completed, the bacterial solution was centrifuged at 4000 rpm and 4 °C for 15 min, and the supernatant was discarded. Subsequently, the collected bacteria were resuspended in 50 mM phosphate buffer with a pH of 7.5 and sonicated in an ice-water bath at 400 W, with each sonication for 3 s and an interval of 7 s. After sonication, the cell lysate was centrifuged at 12,000 rpm and 4 °C for 30 min to remove cell debris. The obtained supernatant was the crude enzyme solution, which was purified by filtration through a 0.22 μm filter membrane. Impurities were washed away with washing buffer (50 mM PBS buffer, 50 mM imidazole, 100 mM NaCl, pH 7.5), and the target protein was eluted with elution buffer (50 mM PBS buffer, 250 mM imidazole, 100 mM NaCl, pH 7.5). The separated target protein was placed in an ultrafiltration centrifugal tube for sufficient desalting and concentration to obtain pure different terminal deoxynucleotidyl transferase mutant proteins.

[0078] Example 4 Characterization of the stability of different terminal deoxynucleotidyl transferase mutants

[0079] To detect the stability of different terminal deoxynucleotidyl transferase mutants, the specific steps are as follows:

[0080] The different terminal deoxynucleotidyl transferase mutant proteins purified in Example 3 were dissolved in PBS buffer to prepare an enzyme solution with a concentration of 0.2 mg / mL. The enzyme solution with a concentration of 0.2 mg / mL was incubated at 56 / 58 / 60 / 62 / 64 / 66 °C for 5 min and then placed on ice for later use. The reaction system included 1 μM primer N, 33 μM ddGTP, 0.25 mM CoCl2, 100 mM NaCl, and 50 mM phosphate buffer (pH 7.5). The enzyme solution of the incubated terminal deoxynucleotidyl transferase mutant at 0.02 mg / mL was reacted at 37 °C for 3 min, and the reaction was analyzed by 20% urea-polyacrylamide gel electrophoresis. The results are as Figures 2 to 3 shown.

[0081] The WT-S / LG and M2-8 / LG proteins purified in Example 3 were dissolved in PBS buffer to prepare an enzyme solution with a concentration of 0.5 mg / mL. The enzyme solution with a concentration of 0.5 mg / mL was incubated at 37 / 58 / 61 °C for 5 min and then placed on ice for later use. The reaction system included 1 μM primer N, 33 μM 3′-ONH2-dGTP, 0.25 mM CoCl2, 100 mM NaCl, and 50 mM phosphate buffer (pH 7.5). The enzyme solution of the incubated enzyme at 0.05 mg / mL was reacted at 37 °C for 10 min, and the reaction was analyzed by 20% urea-polyacrylamide gel electrophoresis. The results are as Figure 4 shown.

[0082] According to the gel electrophoresis images as Figures 2 to 3 shown, SHST analysis was used for gray-scale extraction, the gray-scale of the extended product and the gray-scale of the unextended band were measured, and the yield was calculated by the ratio of the gray-scale of the extended product to the sum of the gray-scales of the extended and unextended bands in this lane. The results are shown in Tables 2, 3, and 4.

[0083] Table 2 Yields of products of terminal deoxynucleotidyl transferase mutants M1-7 at different temperatures

[0084]

[0085] Table 3 Yields of products of terminal deoxynucleotidyl transferase mutants M1-1 to M1-6 at different temperatures

[0086]

[0087]

[0088] Table 4 Yields of products of terminal deoxynucleotidyl transferase mutants M2-2 to M2-8 at different temperatures

[0089]

[0090] Example 5 Unfolding Temperature T of Different Terminal Deoxynucleotidyl Transferase Mutants m Determination of

[0091] The unfolding temperature T of the mutants was determined using a protein thermal stability analyzer (NanoTemper, USA). m A protein sample with a concentration of 0.5 mg / mL was loaded into a quartz capillary, the power was set to 10%, the scanning rate was 60 °C / h, and the scanning range was 20-90 °C.

[0092] Unfolding temperature T of different terminal deoxynucleotidyl transferase mutants m As shown in Table 5.

[0093] Table 5 Terminal deoxynucleotidyl transferase unfolding temperature T m

[0094]

[0095] Depend on Figure 2 , Figure 3 As can be seen from Table 5, M1, M2, M1-4, M1-5, M1-6, M2-2, M2-3, M2-4, M2-5, M2-6, and M2-8 have higher unfolding temperatures than the truncated terminal deoxynucleotidyl transferase, indicating that they have higher thermal stability.

[0096] Example 6 Application of thermostable terminal deoxynucleotidyl transferase in DNA synthesis

[0097] Studies have shown (Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase | ACS Catalysis) that the R335L / K337G combined mutation of WT-L can increase the activity of 3′-ONH2-dNTP. 3′-ONH2 can block the continuous addition of nucleotides, and then remove 3′-ONH2 by NaNO2 for unblocking, which can achieve controlled addition of nucleotides. In Example 1, the N-terminal Loop region of WT-L was truncated, so the 335th and 337th positions of WT-L are the 189th and 191st positions of different mutants obtained later in the example.

[0098] In this example, the M2-8 / LG combined mutant obtained in Example 1 was subjected to DNA extension at high temperature. The specific steps are as follows:

[0099] (1) Construction of WT-S / LG and M2-8 / LG

[0100] Table 6 Construction of WT-S / LG and M2-8 / LG

[0101]

[0102] The primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0103] PCR amplification system:

[0104]

[0105] PCR amplification conditions:

[0106] 1) Pre-denaturation: 98°C for 3 min;

[0107] 2) Denaturation: 98°C for 10 s; Annealing: 60°C for 15 s; Extension: 72°C for 1 min; A total of 30 cycles;

[0108] 3) Post-extension: 72°C for 5 min;

[0109] 4) Store at 4°C.

[0110] Transformation and verification:

[0111] The above PCR products were directly transformed into E. coli BL21(DE3) competent cells by heat shock method. After correct sequencing verification, the truncated terminal deoxynucleotidyl transferase strains E. coli BL21(DE3)-TdT-M2-6 / LG and E. coli BL21(DE3)-TdT-WT-S / LG were obtained.

[0112] (2) Purify M2-8 / LG protein and WT-S / LG protein in the manner of Example 3, dissolve them in PBS buffer to prepare an enzyme solution with a concentration of 0.7 mg / mL. Incubate the BIO-52 primer with magnetic beads at 37°C for 30 min. The reaction system includes the primer, 150 μM 3′-ONH2-dNTP, 0.25 mM CoCl2, 100 mM NaCl and 50 mM phosphate buffer (pH 7.5). Incubate the 0.07 mg / mL enzyme solution after incubation at 55°C for 30 min; After the reaction, incubate with 700 mM NaNO2 for 10 min. Repeat the above process for stepwise extension of nucleotides, and analyze the reaction situation by 20% urea-polyacrylamide gel electrophoresis.

[0113] The results are shown in Figure 5 A and Figure 5 B, where Figure 5 A is the extension effect diagram of M2-8 / LG, Figure 5 B is the extension effect diagram of WT-S / LG.

Claims

1. A terminal deoxynucleotidyl transferase mutant with high thermal stability, characterized in that The mutation form is one of the following: (1) obtained by subjecting the amino acid sequence shown in SEQ ID NO. 4 to G314N / E361P / Q232I / I183M / I328L mutations; (2) obtained by subjecting the amino acid sequence shown in SEQ ID NO. 4 to G314N / E361P / Q232I / I183M / I328L / N31Y mutations; (3) obtained by subjecting the amino acid sequence shown in SEQ ID NO. 4 to G314N / E361P / Q232I / I183M / I328L / N31Y / E275K / D76R mutations; (4) The amino acid sequence shown in SEQ ID NO.4 was mutated to G314N / E361P / Q232I / I183M / I328L / N31Y / E275K / D76R / R311K / I288V / C271Y / T174A / K91E / F297Y / E92R / E278D / I294V, and the amino acid sequence is shown in SEQ ID NO.

5.

2. A terminal deoxynucleotidyl transferase mutant, characterized in that It was obtained by performing R189L / K191G on the amino acid sequence shown in SEQ ID NO.

5.

3. A gene encoding the terminal deoxynucleotidyl transferase mutant according to any one of claims 1 to 2.

4. A recombinant vector, characterized in that: Comprising the gene as claimed in claim 3.

5. A genetically engineered bacterium, characterized in that: Comprising the gene as claimed in claim 3, or comprising the recombinant vector as claimed in claim 4.

6. Use of the terminal deoxynucleotidyl transferase mutant according to any one of claims 1 to 2 in enzymatic DNA synthesis.

7. A method for enzymatically synthesizing DNA, characterized in that: Using deoxyribonucleotide as substrate, DNA is synthesized under the catalysis of the terminal deoxynucleotidyl transferase mutant as claimed in claim 1.

8. The method for enzymatically synthesizing DNA according to claim 7, characterized in that: The 3' end of the deoxyribonucleotide is modified with a reversible blocking group; the terminal deoxynucleotidyl transferase mutant is mutated to improve the activity of binding to the deoxyribonucleotide modified with the reversible blocking group at the 3' end.

9. A method for enzymatically synthesizing DNA, characterized in that: DNA is synthesized under the catalysis of the terminal deoxynucleotidyl transferase mutant described in claim 2 using deoxyribonucleotides modified with 3'-end oxygen amino groups as substrates.

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