Terminal deoxynucleotidyl transferase and its mutants and uses

Abstract

The application discloses a terminal deoxynucleotidyl transferase and a mutant and application thereof. The amino acid sequence of the terminal deoxynucleotidyl transferase (VuTdT-WT) is shown in SEQ ID NO. 1, and the amino acid sequences of the mutant are shown in SEQ ID NO. 3-8. The application compares the activity of VuTdT-WT with that of a reported and commercially available TdT enzyme, and finds that VuTdT-WT shows higher enzymatic activity when acting on natural bases. The application selects VuTdT-WT as an initial enzyme modification starting point, determines the modification direction and the determined modification site of the enzyme through molecular modification, and improves the catalytic activity of the TdT.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a terminal deoxynucleotidyl transferase, its mutants, and their applications. Background Technology

[0002] DNA synthesis is one of the most fundamental and widely used tools in life sciences, and a key enabling technology for synthetic biology. Currently, the phosphoramide solid-phase oligonucleotide synthesis method (phosphoramide method) still dominates. The phosphoramide method uses a phosphoramide monomer with dimethoxytriphenylmethyl (DMT) as a protecting group to achieve the linkage of adjacent nucleotides through the synthesis of a 5'→3' phosphodiester bond. However, this traditional chemical synthesis method still faces several challenges, such as the bottleneck of insufficient synthesis length (generally limited to within 200 nucleotides, theoretically not exceeding 300 nucleotides), low coupling efficiency, high error rate, and the use of large amounts of organic reagents, leading to resource waste. With the continued rapid growth in the scale of DNA synthesis, the hazards, cost burden, and environmental burden of toxic chemicals are becoming increasingly prominent.

[0003] Compared with chemical synthesis, enzymatic oligonucleotide synthesis has several advantages: (1) Enzymatic synthesis reactions are carried out under hydrated and mild conditions. In addition, due to the specificity of enzymes, enzymatic synthesis can reduce the formation of byproducts and damage to oligonucleotides, thus enabling the direct synthesis of longer oligonucleotides; (2) Synthesis can start from natural DNA (i.e., DNA without protecting groups); and (3) Protein engineering (such as directed evolution or rational protein design) can be used to optimize the system, which cannot be achieved by simply using organic chemistry. At present, the core of enzymatic DNA synthesis and various controllable enzymatic DNA synthesis technologies developed based on terminal deoxynucleotidyl transferases still requires highly active terminal deoxynucleotidyl transferases as a foundation. Therefore, it is necessary to develop highly active terminal deoxynucleotidyl transferases. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a terminal deoxynucleotidyltransferase (TdT) with high catalytic activity derived from the wombat (Vombatus ursinus) and its mutants. This TdT enzyme and its mutants exhibit higher catalytic activity compared to existing TdT enzymes.

[0005] The first objective of this invention is to provide a terminal deoxynucleotidyl transferase (VuTdT-WT) comprising: (a) a terminal deoxynucleotidyl transferase having the amino acid sequence SEQ ID NO:1;

[0006] (b) An enzyme that has the terminal deoxynucleotidyltransferase activity of (a) obtained by substitution, deletion or addition of one or more amino acids to the amino acids in (a).

[0007] A second objective of this invention is to provide a gene encoding the aforementioned terminal deoxynucleotidyl transferase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] A third objective of the present invention is to provide a terminal deoxynucleotidyl transferase mutant obtained by mutating amino acid residues or functionally equivalent residues at positions 476 and / or 487 and / or 343 of the amino acid sequence shown in SEQ ID NO.1.

[0009] Preferably, the amino acid sequence of the mutant is as shown in any one of SEQ ID NO.3-8.

[0010] The fourth objective of this invention is to provide a recombinant vector or recombinant bacterium containing the coding gene of the aforementioned terminal deoxynucleotidyl transferase or its mutant.

[0011] A fifth objective of this invention is to provide the application of the aforementioned terminal deoxynucleotidyl transferase or its mutants, or recombinant vectors or recombinant bacteria containing its encoding gene, in catalyzing DNA synthesis reactions.

[0012] Specifically, it provides the application of the aforementioned terminal deoxynucleotidyl transferase or its mutants, or recombinant vectors or recombinant bacteria containing its encoding gene, in catalyzing the addition of deoxynucleotide repeats to the 3' hydroxyl end of oligonucleotides, single-stranded DNA, or double-stranded DNA.

[0013] A sixth object of the present invention is to provide a method for DNA synthesis, the method comprising the step of reacting the above-mentioned terminal deoxynucleotidyl transferase or a mutant thereof with nucleic acid primers and nucleotides.

[0014] A seventh object of the present invention is to provide a composition for catalyzing DNA synthesis reactions, the composition comprising the aforementioned terminal deoxynucleotidyl transferase and / or its mutants.

[0015] The present invention has the following beneficial effects:

[0016] This invention compares the activities of VuTdT-WT (wild-type terminal deoxynucleotidyl transferase) with those of reported, commercially available TdT enzymes. It was found that VuTdT-WT exhibits higher enzyme activity when interacting with natural bases. This invention selects VuTdT-WT as the initial starting point for enzyme modification and determines the direction and specific sites of enzyme modification through molecular modification in order to improve the catalytic activity of the enzyme. Attached Figure Description

[0017] Figure 1 This is an SDS-PAGE image of terminal deoxynucleotidyl transferase.

[0018] Figure 2 PAGE gel image of denatured urea to show the activity of terminal deoxynucleotidyl transferase reaction. Detailed Implementation

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] Unless otherwise specified, the experimental reagents used in the following embodiments of the present invention are all conventional biochemical reagents. Unless otherwise specified, the experimental methods and detection methods involved in the following embodiments are all conventional experimental methods and detection methods already existing in the prior art.

[0021] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0022] The recombinant plasmids and genetically engineered bacteria in the following embodiments of the present invention were all constructed using conventional methods in the art.

[0023] Example 1: Preparation of wild-type terminal deoxynucleotidyl transferase VuTdT-WT and its mutants

[0024] 1. Preparation of wild-type terminal deoxynucleotidyl transferase VuTdT-WT:

[0025] The amino acid sequence of VuTdT-WT is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0026] The gene sequence of VuTdT-WT as shown in SEQ ID NO.2 was cloned into the pET28a plasmid with BamHI / XhoI restriction sites to obtain the recombinant plasmid pET28a-VuTdT-WT. Then, the recombinant plasmid pET28a-VuTdT-WT was introduced into E. coli BL21(DE3) to construct the genetically engineered bacterium E. coli BL21(DE3) / pET28a-VuTdT-WT.

[0027] The constructed genetically engineered strain E. coli BL21(DE3) / pET28a-VuTd-WT was cultured and inoculated into LB medium containing kanamycin (50 μg / mL). The culture was incubated at 37°C and 200 rpm. The absorbance (OD) of the bacterial culture was measured... 600When the β-hydroxyl content reaches 0.6-0.8, 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) is added for induction at 16°C for 20 hours. After culture, the cells are centrifuged to obtain the genetically engineered bacterial cells. The genetically engineered bacterial cells are homogenized and purified by nickel column affinity chromatography to obtain the purified terminal deoxynucleotidyl transferase VuTdT. Figure 1 ).

[0028] >SEQ ID NO.1

[0029] MHRIRTTESDHGKKRQKKMNALISSTIYEIKFHELVLFILEKKMGATRRTFLMDLARKKGFRVENELSNSVTHIVAENNSGSDVLAWLRAHKMEKTEQFELLDVSWLIECMKAGKPVDTKGKYQLVESG VGSINPDPHAGTLKILPPAMKAISQYACQRRTTLNNHNQRFTDAFEILAKNYEFRENPVLCLTFLRATSVLKCLPFAIVSMKDTEGLPWIGDEVKGIMEAIIEEGESLEVQAVLNDERYQSFKLFTSVFG VGIKTSEKWYRMGFRTLREIRSDKSLRFTKMQEAGFRYYDDIIACVSKAEADAVSLLVKEAVWTFLPDALVTTTGGFRRRKEFGHDVDFLITSPGAEKEQEDQLLQRVTNLWEKQGLLLYCDLIESTFED LKLPSRKVDALDHFQKCFLILKLYHRRGDMSKWEMPAGSNVLEAKNWKAIRVDLVVCPYDRYAYALLGWSGSRQFERDLRRYATHEKKMMLDNHALYDKTKRIFLEAKSEEEIFEHLGLEYIEPWERNA.

[0030] >SEQ ID NO.2

[0031]

[0032] 2. Preparation of VuTdT mutant:

[0033] The VuTdT mutant was prepared by mutating amino acid residues at positions 476 and / or 487 and / or 343 of the amino acid sequence shown in SEQ ID NO.1. The specific mutation details are shown in Table 1 below.

[0034] Table 1. Mutants included in this invention

[0035] serial number Mutation Combination corresponding amino acid sequence Mut1 K476Y SEQ ID NO.3 Mut2 D487W SEQ ID NO.4 Mut3 G343R SEQ ID NO.5 Mut4 K476Y-D487W SEQ ID NO.6 Mut5 K476Y-G343R SEQ ID NO.7 Mut6 D487W-G343R SEQ ID NO.8

[0036] The preparation steps for the above mutants Mut1-Mut6 are the same as in step 1.

[0037] >SEQ ID NO.3

[0038] MHRIRTTESDHGKKRQKKMNALISSTIYEIKFHELVLFILEKKMGATRRTFLMDLARKKGFRVENELSNSVTHIVAENNSGSDVLAWLRAHKMEKTEQFELLDVSWLIECMKAGKPVDTKGKYQLVESG VGSINPDPHAGTLKILPPAMKAISQYACQRRTTLNNHNQRFTDAFEILAKNYEFRENPVLCLTFLRATSVLKCLPFAIVSMKDTEGLPWIGDEVKGIMEAIIEEGESLEVQAVLNDERYQSFKLFTSVFG VGIKTSEKWYRMGFRTLREIRSDKSLRFTKMQEAGFRYYDDIIACVSKAEADAVSLLVKEAVWTFLPDALVTTTGGFRRRKEFGHDVDFLITSPGAEKEQEDQLLQRVTNLWEKQGLLLYCDLIESTFED LKLPSRKVDALDHFQKCFLILKLYHRRGDMSKWEMPAGSNVLEAKNWKAIRVDLVVCPYDRYAYALLGWSGSRQFERDLRRYATHEYKMMLDNHALYDKTKRIFLEAKSEEEIFEHLGLEYIEPWERNA.

[0039] >SEQ ID NO.4

[0040] MHRIRTTESDHGKKRQKKMNALISSTIYEIKFHELVLFILEKKMGATRRTFLMDLARKKGFRVENELSNSVTHIVAENNSGSDVLAWLRAHKMEKTEQFELLDVSWLIECMKAGKPVDTKGKYQLVESGVGSINPDPHAGTLKILPPAMKAISQYACQRRTTLNNHNQRFTDAFEIAKNYEFRENPVLCLTFLRATSVLKCLPFAIVSMKDTEGLPWIGDEVKGIMEAIIEEGESLEVQAVLNDERYQSFKLFTSVFGVGIKTSEKWYRMGFRTLREIRSDKSLRFTKMQEAGFRYYDDIIACVSKAEADAVSLLVKEAVWTFLPDALVTTTGGFRRRKEFGHDVDFLITSPGAEKEQEDQLLQRVTNLWEKQGLLLYCDLIESTFEDLKLPSRKVDALDHFQKCFLILKLYHRRGDMSKWEMPAGSNVLEAKNWKAIRVDLVVCPYDRYAYALLGWSGSRQFERDLRRYATHEKKMMLDNHALYWKTKRIFLEAKSEEEIFEHLGLEYIEPWERNA.

[0041] >SEQ ID NO.5

[0042] MHRIRTTESDHGKKRQKKMNALISSTIYEIKFHELVLFILEKKMGATRRTFLMDLARKKGFRVENELSNSVTHIVAENNSGSDVLAWLRAHKMEKTEQFELLDVSWLIECMKAGKPVDTKGKYQLVESGVGSINPDPHAGTLKILPPAMKAISQYACQRRTTLNNHNQRFTDAFEILAKNYEFRENPVLCLTFLRATSVLKCLPFAIVSMKDTEGLPWIGDEVKGIMEAIIEEGESLEVQAVLNDERYQSFKLFTSVFGVGIKTSEKWYRMGFRTLREIRSDKSLRFTKMQEAGFRYYDDIIACVSKAEADAVSLLVKEAVWTFLPDALVTTTGGFRRRKEFRHDVDFLITSPGAEKEQEDQLLQRVTNLWEKQGLLLYCDLIESTFEDLKLPSRKVDALDHFQKCFLILKLYHRRGDMSKWEMPAGSNVLEAKNWKAIRVDLVVCPYDRYAYALLGWSGSRQFERDLRRYATHEKKMMLDNHALYDKTKRIFLEAKSEEIFEHLGLEYIEPWERNA.

[0043] >SEQ ID NO.6

[0044] MHRIRTTESDHGKKRQKKMNALISSTIYEIKFHELVLFILEKKMGATRRTFLMDLARKKGFRVENELSNSVTHIVAENNSGSDVLAWLRAHKMEKTEQFELLDVSWLIECMKAGKPVDTKGKYQLVESGVGSINPDPHAGTLKILPPAMKAISQYACQRRTTLNNHNQRFTDAFEILAKNYEFRENPVLCLTFLRATSVLKCLPFAIVSMKDTEGLPWIGDEVKGIMEAIIEEGESLEVQAVLNDERYQSFKLFTSVFGVGIKTSEKWYRMGFRTLREIRSDKSLRFTKMQEAGFRYYDDIIACVSKAEADAVSLLVKEAVWTFLPDALVTTTGGFRRRKEFGHDVDFLITSPGAEKEQEDQLLQRVTNLWEKQGLLLYCDLIESTFEDLKLPSRKVDALDHFQKCFLILKLYHRRGDMSKWEMPAGSNVLEAKNWKAIRVDLVVCPYDRYAYALLGWSGSRQFERDLRRYATHEYKMMLDNHALYWKTKRIFLEAKSEEIFEHLGLEYIEPWERNA.

[0045] >SEQ ID NO.7

[0046] MHRIRTTESDHGKKRQKKMNALISSTIYEIKFHELVLFILEKKMGATRRTFLMDLARKKGFRVENELSNSVTHIVAENNSGSDVLAWLRAHKMEKTEQFELLDVSWLIECMKAGKPVDTKGKYQLVESGVGSINPDPHAGTLKILPPAMKAISQYACQRRTTLNNHNQRFTDAFEILAKNYEFRENPVLCLTFLRATSVLKCLPFAIVSMKDTEGLPWIGDEVKGIMEAIIEEGESLEVQAVLNDERYQSFKLFTSVFGVGIKTSEKWYRMGFRTLREIRSDKSLRFTKMQEAGFRYYDDIIACVSKAEADAVSLLVKEAVWTFLPDALVTTTGGFRRRKEFRHDVDFLITSPGAEKEQEDQLLQRVTNLWEKQGLLLYCDLIESTFEDLKLPSRKVDALDHFQKCFLILKLYHRRGDMSKWEMPAGSNVLEAKNWKAIRVDLVVCPYDRYAYALLGWSGSRQFERDLRRYATHEYKMMLDNHALYDKTKRIFLEAKSEEIFEHLGLEYIEPWERNA.

[0047] >SEQ ID NO.8

[0048] MHRIRTTESDHGKKRQKKMNALISSTIYEIKFHELVLFILEKKMGATRRTFLMDLARKKGFRVENELSNSVTHIVAENNSGSDVLAWLRAHKMEKTEQFELLDVSWLIECMKAGKPVDTKGKYQLVESG VGSINPDPHAGTLKILPPAMKAISQYACQRRTTLNNHNQRFTDAFEILAKNYEFRENPVLCLTFLRATSVLKCLPFAIVSMKDTEGLPWIGDEVKGIMEAIIEEGESLEVQAVLNDERYQSFKLFTSVFG VGIKTSEKWYRMGFRTLREIRSDKSLRFTKMQEAGFRYYDDIIACVSKAEADAVSLLVKEAVWTFLPDALVTTTGGFRRRKEFRHDVDFLITSPGAEKEQEDQLLQRVTNLWEKQGLLLYCDLIESTFED LKLPSRKVDALDHFQKCFLILKLYHRRGDMSKWEMPAGSNVLEAKNWKAIRVDLVVCPYDRYAYALLGWSGSRQFERDLRRYATHEKKMMLDNHALYWKTKRIFLEAKSEEEIFEHLGLEYIEPWERNA.

[0049] Example 2: Catalytic activity test of VuTdT

[0050] The activity of VuTdT wild-type was detected using commercially available NEB TdT terminal transferase (catalog number: M0315L) and TdT from white-throated sparrow (ZaTdT) as controls. A reaction preparation solution containing 1 μM oligonucleotide primers, 0.1 mM deoxyribonucleotides, and 0.25 mM CoCl2 was prepared using Tris-HCl, pH 8.0 buffer. The reaction was carried out in a 30°C metal bath, followed by inactivation at 95°C for 5 min. The supernatant was collected for detection of VuTdT and TdT from the two different sources. 20% urea-polyacrylamide gel (denaturing PAGE) was used for detection, and the results after 1×SYBR Gold staining are shown below. Figure 2 As shown.

[0051] Depend on Figure 2 As can be seen, the single nucleotide incorporation efficiency of VuTdT in the examples is better than that of ZaTdT and NEB TdT, exhibiting higher catalytic efficiency, and can efficiently synthesize nucleic acid molecules without template strand.

[0052] Example 3: Catalytic rate test of VuTdT or its mutant

[0053] DNA synthase (VuTdT or its mutant) was added to Tris-HCl, pH 8.0 buffer to prepare a reaction solution containing 1 μM oligonucleotide primers, 0.1 mM deoxyribonucleotides, and 0.25 mM CoCl2. The reaction was carried out in a 30°C metal bath, followed by inactivation at 95°C for 5 min. The supernatant was then collected for detection of the catalytic rate of VuTdT and its mutants. Specifically, a pyrophosphate fluorescent probe kit was used to test the catalytic rate of VuTdT and its mutants. 50 μL of the reaction solution and 50 μL of the pyrophosphate fluorescent probe solution were added to a black 96-well microplate. After incubation at room temperature for 10 min, the light intensity at wavelengths of 316 nm and 456 nm was read using a multi-mode microplate reader in luminescence mode. Monitoring was performed continuously for 2 min, and the average luminescence value during this time period was calculated. The catalytic efficiency of VuTdT or its mutants is shown in Table 2 below.

[0054] Table 2 Catalytic efficiency of VuTdT or its mutants

[0055] enzymes Catalytic efficiency mM / min VuTdT 6.30 Mut1 20.5 Mut2 17.89 Mut3 156.8 Mut4 25.84 Mut5 78.35 Mut6 291.78

[0056] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A terminal deoxynucleotidyl transferase mutant, characterized in that, The amino acid sequence of the mutant is shown in any one of SEQ ID NO.3-8.

2. The encoding gene of the terminal deoxynucleotidyl transferase mutant according to claim 1.

3. A recombinant vector or recombinant bacterium containing the coding gene of the terminal deoxynucleotidyl transferase mutant as described in claim 2.

4. The application of the terminal deoxynucleotidyl transferase mutant of claim 1 or the recombinant vector or recombinant bacteria of claim 3 in catalyzing DNA synthesis reactions.

5. The application according to claim 4, characterized in that, It is used in the catalytic addition of deoxynucleotide repeats to the 3' hydroxyl end of oligonucleotides, single-stranded DNA, or double-stranded DNA.

6. A method for DNA synthesis, characterized in that, The method includes the step of reacting the terminal deoxynucleotidyl transferase mutant of claim 1 with nucleic acid primers and nucleotides.

7. A composition for catalyzing DNA synthesis reactions, characterized in that, It includes the terminal deoxynucleotidyl transferase mutant as described in claim 1.

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