Terminal deoxynucleotidyl transferase mutant and application thereof

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly relates to a terminal deoxynucleotidyl transferase mutant and application thereof. According to the application, the TdT structure of a wild type bird is analyzed, and amino acid sites of glutamic acid (E) at the 47th site, glutamic acid (E) at the 54th site, lysine (K) at the 158th site, arginine (R) at the 210th site, lysine (K) at the 212th site, asparagine (N) at the 213th site, isoleucine (I) at the 214th site, phenylalanine (F) at the 273th site and arginine (R) at the 332nd site, which influence the activity of the enzyme, are specifically designed; the terminal deoxynucleotidyl transferase mutant designed by the invention can effectively improve the activity of enzyme.

Description

Technical Field

[0001] This application belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a terminal deoxynucleotidyl transferase mutant and its application. Background Art

[0002] DNA synthesis technologies mainly include chemical methods and biological methods. Among them, the chemical method (especially the solid-phase phosphoramidite triester synthesis method) is the most mature and widely used, while the biological method has emerged abroad but is still in the principle verification stage.

[0003] With the increase in the length of oligonucleotide synthesis by the chemical method, the error rate shows an upward trend, and the product yield also decreases significantly. Moreover, a large amount of chemical reagents need to be used during the synthesis process, involving strong acids and strong oxidants, and the waste liquid and waste gas generated cause serious environmental pollution, resulting in high subsequent treatment costs.

[0004] In recent years, experts and scholars have turned their attention to the biological synthesis method that does not rely on chemical reagents. The DNA synthesis technology by the bioenzymatic method is usually carried out in an aqueous environment, which can effectively avoid the above problems and is expected to synthesize longer DNA molecules at a lower cost.

[0005] The bioenzymatic method includes biological synthesis technologies such as the enzymatic method of terminal deoxynucleotidyl transferase (TdT), the coupling method, the mixed enzyme method, and metal ion regulation. TdT and some DNA polymerases can directly catalyze the synthesis of DNA strands without relying on existing DNA template molecules, and combined with in vivo assembly methods such as homologous recombination, the length and accuracy of oligonucleotide synthesis can be improved by several orders of magnitude, greatly enhancing the ability of design and construction using synthetic biology. Compared with the chemical method of DNA synthesis, the bioenzymatic method has great prospects and is expected to create significant value in terms of synthesis length and yield.

[0006] TdT is a non-template-dependent enzyme that usually extends DNA strands randomly and can add 4 natural bases to the 3'-end of the DNA strand. By chemically synthesizing nucleotide monomers with reversible terminating groups, and then using the TdT enzyme to continuously add bases to the end of the synthesized sequence, only one target single base can be extended each time. Subsequently, the terminating group is removed and the synthesis of the next target base begins. A total of two steps can complete one round of base incorporation.

[0007] However, the activity of the terminal transferase mutant obtained based on traditional technologies still cannot fully meet the actual requirements. Summary of the Invention

[0008] Based on this, an embodiment of this application provides a mutant of terminal deoxynucleotidyl transferase, which can effectively improve the enzyme activity.

[0009] On the one hand, the present application provides a mutant of terminal deoxynucleotidyl transferase. The amino acid sequence of the mutant of terminal deoxynucleotidyl transferase has one or more amino acid substitutions compared with the sequence shown in SEQ ID NO: 1, and the substitution sites include at least one of the 47th, 54th, 158th, 210th, 212th, 213th, 214th, 273rd and 332nd positions.

[0010] In one embodiment, the amino acid substitution includes at least one of E47T, E47S, E54S, K158T, R210L, K212G, N213E, N213Q, I214T, I214M, F273L and R332K.

[0011] In one embodiment, the amino acid substitution includes one of the following 12 combinations:

[0012] Combination (1): R210L and K212G;

[0013] Combination (2): R210L, K212G and N213E;

[0014] Combination (3): R210L, K212G, N213Q and I214T;

[0015] Combination (4): R210L, K212G, N213Q and I214M;

[0016] Combination (5): R210L, K212G and E47T;

[0017] Combination (6): R210L, K212G and E54S;

[0018] Combination (7): R210L, K212G and F273L;

[0019] Combination (8): R210L, K212G and R332K;

[0020] Combination (9): R210L, K212G, E47S and E54S;

[0021] Combination (10): R210L, K212G, E47T, E54S and R332K;

[0022] Combination (11): R210L, K212G, E47S, E54S and R332K;

[0023] Combination (12): R210L, K212G and K158T.

[0024] The present application also provides a nucleic acid molecule encoding the mutant of terminal deoxynucleotidyl transferase.

[0025] The present application also provides an expression vector comprising the nucleic acid molecule.

[0026] In one embodiment, it includes a plasmid.

[0027] In one embodiment, the plasmid includes pET-28a plasmid.

[0028] The present application also provides a host cell comprising the nucleic acid molecule or the expression vector.

[0029] In one embodiment, the host cell includes Escherichia coli cells.

[0030] In one embodiment, the Escherichia coli includes E.coli BL21(DE3).

[0031] The present application also provides a method for producing a mutant of terminal deoxynucleotidyl transferase, which comprises the following steps: culturing the host cell; and isolating the mutant from the obtained culture.

[0032] The present application also provides a nucleic acid fragment synthesis kit, which includes the mutant, the nucleic acid molecule, the expression vector or the host cell.

[0033] In one embodiment, it further includes other nucleic acid fragment synthesis reagents.

[0034] In one embodiment, the other nucleic acid fragment synthesis reagents include modified dNTP, Co 2+ 、Na + 、and one or more of reaction buffers.

[0035] In one embodiment, the reaction buffer includes 45 mM - 55 mM Tris-HCl buffer with a pH value of 7.0 - 7.4.

[0036] Compared with the traditional technology, the beneficial effects of the present application include:

[0037] By analyzing the structure of wild-type avian TdT, specific designs were made for the amino acid sites that affect the enzyme activity, namely Glutamic acid (E) at position 47, Glutamic acid (E) at position 54, Lysine (K) at position 158, Arginine (R) at position 210, Lysine (K) at position 212, Asparagine (N) at position 213, Isoleucine (I) at position 214, Phenylalanine (F) at position 273, and Arginine (R) at position 332. The mutant of terminal deoxynucleotidyl transferase designed through this application can effectively improve the enzyme activity. Brief Description of the Drawings

[0038] To more clearly illustrate the technical solutions in the embodiments of this application and to more fully understand this application and its beneficial effects, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of this application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0039] Figure 1 SDS-PAGE detection chart of TdT4 / 95 / 103 / 104 / 113 / 117 / 133 / 164 / 228 / 230 / 231 / T4CC8 mutant protein;

[0040] Figure 2 SDS-PAGE detection chart of the incorporation effect of TdT4 / 113 / 117 / 133 / 164 / 228 / 230 / 231 / 95 / T4CC8 on the catalytic modification substrate 3’-ONH 2 -dATP;

[0041] Figure 3 SDS-PAGE detection chart of the incorporation of TdT228 / 231 / 103 / 104 on the catalytic modification substrate 3’-ONH 2 -dATP;

[0042] Figure 4 SDS-PAGE detection chart of the incorporation of TdT104 / 228 on four modified substrates. Detailed Embodiments

[0043] The present application will be further described in detail below in conjunction with the embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the disclosure of the present application more thoroughly and comprehensively understood. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0044] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs.

[0045] The term

[0046] All documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. Unless it conflicts with the purpose and / or technical solution of the present application, the cited documents involved in the present application are incorporated by reference in their entirety and for all purposes. When the present application involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When the present application involves cited documents, the examples and preferred methods of the relevant technical features cited can also be incorporated as references into the present application, but only to the extent that the present application can be implemented. It should be understood that when the cited content conflicts with the description in the present application, the present application shall prevail or be modified adaptively according to the description in the present application.

[0047] Unless otherwise specified or there is a contradiction, the terms or phrases used herein have the following meanings:

[0048] The term "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are used to connect at least three items, it should be understood that in the present application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR".

[0049] The term "mutation" refers to the deletion, addition, or substitution of amino acid residues in the amino acid sequence of a protein or polypeptide as compared to the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, the substitution of an amino acid at a specific position in a protein sequence is denoted using annotations such as R210L, which means that the arginine (R) residue at position 210 of the amino acid sequence of the reference protein is replaced by a lysine (L) residue (in the mutant of the reference protein).

[0050] The term "SDS-PAGE", sodium dodecyl sulfate polyacrylamide gel electrophoresis, is one of the most commonly used protein expression analysis techniques in polyacrylamide gel electrophoresis. The principle of this technique is to separate proteins in an electrophoresis gel according to their different molecular weights in the sample. In the experiment of expressing and purifying foreign proteins in Escherichia coli, SDS-PAGE is an essential operation, which is usually used to detect the expression of proteins (expression level, expression distribution), and analyze the purity of the target protein, etc.

[0051] The term "qPCR", Quantitative Real-time PCR, is a method in DNA amplification reactions that measures the total amount of products after each polymerase chain reaction (PCR) cycle using a fluorescent chemical substance. It is a method for quantitatively analyzing specific DNA sequences in a test sample by an internal reference or external reference method.

[0052] Real-time PCR is used to detect the PCR process in real time through fluorescent signals during the PCR amplification process. Since there is a linear relationship between the Ct value of the template and the initial copy number of the template during the exponential phase of PCR amplification, it becomes the basis for quantification.

[0053] The term "vector" refers to a nucleic acid molecule that can transport or transfer foreign nucleic acid molecules. This term encompasses both expression vectors and transcription vectors. The term "expression vector" refers to a vector that can express an insert in a target cell and usually contains control sequences (such as enhancer, promoter, and terminator sequences) that drive the expression of the insert. The term "transcription vector" refers to a vector that can be transcribed but not translated. Transcription vectors are used to amplify their inserts. Foreign nucleic acid molecules are called "inserts" or "transgenes". Vectors usually consist of an insert and a larger sequence that serves as the backbone of the vector. Based on the structure or source of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors (such as λ phage), viral vectors (such as adenovirus vectors), and artificial chromosomes.

[0054] The term "host cell" refers to a cell in which a vector can be propagated and its DNA or RNA can be expressed. Such a cell can be prokaryotic or eukaryotic, and a recipient cell is also called a host cell. Recipient cells include prokaryotic recipient cells (mainly Escherichia coli), eukaryotic recipient cells (mainly Saccharomyces cerevisiae), animal cells, and insect cells (which are actually also eukaryotic recipient cells). Among prokaryotic recipient cells, the most commonly used host cell is Escherichia coli.

[0055] On the one hand, the present application provides a mutant of terminal deoxynucleotidyl transferase. The amino acid sequence of the mutant of terminal deoxynucleotidyl transferase has one or more amino acid substitutions compared with the sequence shown in SEQ ID NO: 1, and the substitution sites include at least one of the 47th, 54th, 158th, 210th, 212th, 213th, 214th, 273rd, and 332nd positions.

[0056] In a specific example, the amino acid substitutions include at least one of E47T, E47S, E54S, K158T, R210L, K212G, N213E, N213Q, I214T, I214M, F273L, and R332K.

[0057] In one of the embodiments, the amino acid substitutions include one of the following 12 combinations:

[0058] Combination (1): R210L and K212G;

[0059] Combination (2): R210L, K212G, and N213E;

[0060] Combination (3): R210L, K212G, N213Q, and I214T;

[0061] Combination (4): R210L, K212G, N213Q, and I214M;

[0062] Combination (5): R210L, K212G, and E47T;

[0063] Combination (6): R210L, K212G, and E54S;

[0064] Combination (7): R210L, K212G, and F273L;

[0065] Combination (8): R210L, K212G, and R332K;

[0066] Combination (9): R210L, K212G, E47S, and E54S;

[0067] Combinations (10): R210L, K212G, E47T, E54S, and R332K;

[0068] Combinations (11): R210L, K212G, E47S, E54S, and R332K;

[0069] Combinations (12): R210L, K212G, and K158T.

[0070] This application also provides a nucleic acid molecule encoding the mutant of terminal deoxynucleotidyl transferase described above.

[0071] This application also provides an expression vector comprising the nucleic acid molecule described above. There is no particular limitation on the vector into which the nucleic acid encoding the mutant of terminal deoxynucleotidyl transferase of this application is inserted, and any vector commonly used in the art can be used. A vector capable of autonomous replication in a host cell or a vector that can be integrated into the host chromosome can be used.

[0072] Optionally, it includes a plasmid. It can be understood that plasmid vectors, phage vectors, viral vectors, etc. are included. As plasmid vectors, plasmids suitable for the host to be used, such as plasmids derived from Escherichia coli (E. coli), plasmids derived from bacteria of the genus Bacillus, or plasmids derived from yeast, are well-known to those skilled in the art, and many plasmid vectors are commercially available. In this application, these known plasmids and plasmids modified from known plasmids can be used. As phage vectors, for example, λ phage can be used. As viral vectors, for example, animal viruses such as retroviruses or vaccinia viruses, or insect viruses such as baculoviruses can be used.

[0073] Further optionally, the plasmid includes but is not limited to the pET-28a plasmid.

[0074] This application also provides a host cell comprising the nucleic acid molecule or the expression vector described above. There is no particular limitation on the method for introducing the expression vector into the host, as long as it can introduce the nucleic acid into the host, and examples thereof include: methods using calcium ions, electroporation, spheroplast method, and lithium acetate method.

[0075] In a specific example, the host cell includes but is not limited to Escherichia coli cells.

[0076] Optionally, the Escherichia coli includes but is not limited to E. coli BL21(DE3).

[0077] This application also provides a method for producing a mutant of terminal deoxynucleotidyl transferase, which includes the following steps: culturing the host cell described above; and isolating the mutant from the resulting culture.

[0078] The present application also provides a nucleic acid fragment synthesis kit, which includes the mutant, the nucleic acid molecule, the expression vector or the host cell described above.

[0079] In a specific example, it further includes other nucleic acid fragment synthesis reagents.

[0080] Optionally, the other nucleic acid fragment synthesis reagents include modified dNTP, Co 2+ , Na + , and one or more of reaction buffers.

[0081] In one embodiment, the reaction buffer includes 45 mM to 55 mM Tris-HCl buffer with a pH value of 7.0 to 7.4. For example, Tris-HCl buffers of 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM with pH values of 7.0, 7.1, 7.2, 7.3, 7.4.

[0082] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present application is preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0083] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0084] Example 1

[0085] I. Experimental Materials

[0086] 1. Experimental Instruments

[0087] PCR instrument, laminar flow hood, shaker, high-throughput non-contact ultrasonic crusher, centrifuge, refrigerated centrifuge, protein purifier, magnetic separator, electrophoresis instrument, electrophoresis tank, gel imager, microwave oven.

[0088] 2. Experimental Consumables

[0089] Pipette, disposable tips, EP tubes, disposable plates, power strips, test tubes, shake flasks, centrifuge tubes, centrifuge cups, protein purification magnetic beads, protein purification columns, nickel fillers.

[0090] 3. Preparation of Experimental Reagents

[0091] The formulation of the fermentation medium is shown in Table 1. The protein purification buffer includes an equilibration buffer and a washing buffer, as specifically shown in Tables 2-4: Table 1

[0092] Component Concentration Tryptone 10 g / L Yeast extract 5 g / L NaCl 10 g / L

[0093] Among them, the formulation of the equilibration buffer is shown in Table 2:

[0094] Table 2

[0095] Component Concentration Tris-HCl pH 7.2 50 mM NaCl 300 mM

[0096] The washing buffer is shown in Table 3:

[0097] Table 3

[0098] Component Concentration Tris-HCl pH 7.2 50 mM NaCl 300 mM Imidazole 50 mM

[0099] The elution buffer is shown in Table 4:

[0100] Table 4

[0101] Component Concentration Tris-HCl pH 7.2 50 mM NaCl 300 mM Imidazole 300 mM Glycerol 10％

[0102] 4. Experimental strains: E. coli top10, E. coli BL21(DE3)

[0103] 5. Experimental vectors: pET series

[0104] II. Specific process

[0105] 1. Gene design and synthesis

[0106] In order to improve the catalytic activity of TdT towards modified substrates, the structure of wild-type avian TdT was analyzed, and the amino acid sites that affect the activity of this enzyme, namely glutamate (E) at position 47, glutamate (E) at position 54, lysine (K) at position 158, arginine (R) at position 210, lysine (K) at position 212, asparagine (N) at position 213, isoleucine (I) at position 214, phenylalanine (F) at position 273, and arginine (R) at position 332, were optimized.

[0107] While maintaining the secondary structure unchanged, glutamate (E) at position 47 can be mutated to threonine (T) / serine (S) (E47T / E47S), glutamate (E) at position 54 can be mutated to serine (S) (E54S), lysine (K) at position 158 can be mutated to threonine (T) (K158T), arginine (R) at position 210 can be mutated to leucine (L) (R210L), lysine (K) at position 212 can be mutated to glycine (G) (K212G), asparagine (N) at position 213 can be mutated to glutamate (E) or glutamine (Q) (N213E / N213Q), isoleucine (I) at position 214 can be mutated to threonine (T) / methionine (M) (I214T / I214M), phenylalanine (F) at position 273 can be mutated to leucine (L) (F273L), and arginine (R) at position 332 can be mutated to lysine (K) (R332K), resulting in the following 12 avian TdT mutants: TdT4, TdT95, TdT103, TdT104, TdT113, TdT117, TdT133, TdT164, TdT228, TdT230, TdT231, T4CC8. The catalytic activities of the TdT mutants were determined.

[0108] The specific sites and corresponding mutants are shown in Table 5 below:

[0109] Table 5

[0110] Serial number Mutation site Sequence TdT 4 R210L + K212G SEQ ID NO.2 TdT 95 R210L + K212G + N213E SEQ ID NO.3 TdT 103 R210L + K212G + N213Q + I214T SEQ ID NO.4 TdT 104 R210L + K212G + N213Q + I214M SEQ ID NO.5 TdT 113 R210L + K212G + E47T SEQ ID NO.6 TdT 117 R210L + K212G + E54S SEQ ID NO.7 TdT 133 R210L + K212G + F273L SEQ ID NO.8 TdT 164 R210L + K212G + R332K SEQ ID NO.9 TdT 228 R210L + K212G + E47S + E54S SEQ ID NO.10 TdT 230 R210L + K212G + E47T + E54S + R332K SEQ ID NO.11 TdT 231 R210L + K212G + E47S + E54S + R332K SEQ ID NO.12 T4CC8 R210L + K212G + K158T SEQ ID NO.13

[0111] After the TdT wild-type and mutants were designed, gene synthesis (synthesized by Nanjing Tsingke Biotechnology Co., Ltd.) and codon optimization were performed and then ligated into the pET-28a plasmid to obtain the expression vectors of the TdT wild-type and mutants.

[0112] 2. Induced expression

[0113] Extract the expression plasmids of the TdT mutants from E. coli top10, and slowly add 5 μl - 10 μl respectively to the competent cells of E. coli BL21(DE3). After mixing, incubate on ice for 30 min, heat shock at 42 °C for 45 s - 90 s, and then incubate on ice for 1 min - 2 min. Add 900 μl of LB and incubate at 37 °C at 220 rpm for 1 h, centrifuge at 5000 rpm for 3 min, leave a little supernatant, pipette and mix well, and spread on the LB plate medium containing kanamycin resistance. Incubate overnight at 37 °C in an inverted position. Pick the above monoclonal colonies and inoculate them into 5 mL of LB medium containing kanamycin resistance, culture overnight at 37 °C at 220 rpm. Inoculate the bacterial solution into 100 mL of LB medium containing kanamycin resistance at an inoculation amount of 1%, culture at 37 °C at 220 rpm until OD 0.6 - 0.8, add IPTG with a final concentration of 0.5 mM, and induce overnight at 16 °C at 120 rpm.

[0114] 3. Protein purification

[0115] (1) Cell disruption

[0116] After the induction of expression was completed, 40 mL of the bacterial solution was centrifuged at 3500 rpm for 15 min. The supernatant was discarded, and the pellet was resuspended in 1.5 mL of equilibration buffer. 1 mM protease inhibitor was added. The cells were lysed using a high-throughput non-contact ultrasonic cell disruptor. The disruption temperature was 4°C, the power was 100%, the intermittent time was 3 s, and the total duration was 60 min. After disruption, the mixture was centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected.

[0117] (2) Preparation and equilibration of magnetic beads

[0118] Take 150 μl of the magnetic bead suspension and place it on a magnetic separator. After the solution becomes clear, aspirate and discard the clear liquid with a pipette. Add 200 μl of equilibration buffer, pipette up and down 5 - 10 times, place it on the magnetic separator, aspirate and discard the clear liquid with a pipette, and repeat the washing 2 times.

[0119] (3) Binding of the target protein to magnetic beads

[0120] Add the centrifuged supernatant to the treated magnetic beads and invert to mix evenly. Incubate with gentle rotation at 40 rpm for 1 h at 4°C, then centrifuge to remove the supernatant. Remove the centrifuge tube from the magnetic separator for washing. Add 400 μl of washing solution to the centrifuge tube, pipette up and down 5 - 10 times with a pipette tip to remove the supernatant, and repeat 1 time. Add 50 μl - 100 μl of elution buffer to the centrifuge tube, incubate with gentle rotation at 40 rpm for 10 min at 4°C, then centrifuge to obtain the target protein.

[0121] (4) SDS-PAGE detection

[0122] Use a 4% - 20% SDS-PAGE precast gel (provided by Hubei Qingke Biotechnology Co., Ltd.) and place it in the electrophoresis tank for gel running. Take different TdT mutant proteins, add Loading buffer and mix evenly. Load 20 μl of the sample and 5 μl of Marker. Run the gel at 160 V for 30 min. Stain with the staining solution by heating for 15 min. After cooling, take a photo using a gel imager. Analyze whether the size of the target protein band is correct with reference to the size of the Marker band.

[0123] (5) Protein concentration determination

[0124] After cleaning with ultrapure water, zero the instrument with elution buffer, and then use the SAM 4000 to measure the concentration of the purified target protein and the 260 / 280 value.

[0125] (6) TdT activity detection

[0126] A. Detection of the activity of the TdT mutant in incorporating and catalyzing the modification substrate for different starting strands at the ends

[0127] Prepare the reaction system on ice. The specific formula is shown in Table 6 below:

[0128] Table 6

[0129] Component Concentration (total volume 25 μl) Initial strand* 1 μM 3’-ONH2-dATP 0.25 mM <![CDATA[CoCl 2 > 0.25 mM NaCl 100 mM Tris-HCl 7.2 50 mM TdT 0.5 mg / ml <![CDATA[H 2 O]]> Make up to 25 μl

[0130] * The starting strands include:

[0131] P1-AA: TTTTTTTTTTTTTTAA, P1-AT: TTTTTTTTTTTTTTAT,

[0132] P1-AG: TTTTTTTTTTTTTTAG, P1-AC: TTTTTTTTTTTTTTAC,

[0133] P1-TA: TTTTTTTTTTTTTTTA, P1-TT: TTTTTTTTTTTTTTT,

[0134] P1-TG: TTTTTTTTTTTTTTTTG, P1-TC: TTTTTTTTTTTTTTTC,

[0135] P1-GA: TTTTTTTTTTTTTTGA, P1-GT: TTTTTTTTTTTTTTGT,

[0136] P1-GG: TTTTTTTTTTTTTTGG, P1-GC: TTTTTTTTTTTTTTGC,

[0137] P1-CA: TTTTTTTTTTTTTTCA, P1-CT: TTTTTTTTTTTTTTCT,

[0138] P1-CG: TTTTTTTTTTTTTTCG, P1-CC: TTTTTTTTTTTTTTCC.

[0139] The reaction conditions are: react at 30 °C for 30 s and heat at 95 °C for 10 min.

[0140] B. Detection of the activity of the TdT mutant in incorporating and catalyzing the modification substrate for different starting strands at the ends (increasing the metal ion concentration)

[0141] Prepare the reaction system on ice. The specific formula is shown in Table 7 below:

[0142] Table 7

[0143] Component Concentration (total volume 25 μl) Initial strand* 1 μM 3’-ONH2-dATP 0.25 mM <![CDATA[CoCl 2 > 2.5 mM NaCl 100 mM Tris-HCl 7.2 50 mM TdT 0.5 mg / ml <![CDATA[H 2 O]]> Make up to 25 μl

[0144] The initial chains include:

[0145] P1-AC: TTTTTTTTTTTTTTAC, P1-TC: TTTTTTTTTTTTTTC,

[0146] P1-GC: TTTTTTTTTTTTTTGC, P1-CA: TTTTTTTTTTTTTTCA,

[0147] P1-CT: TTTTTTTTTTTTTTCT, P1-CG: TTTTTTTTTTTTTCG,

[0148] P1-CC: TTTTTTTTTTTTTTCC.

[0149] Reaction conditions: React at 30 °C for 30 s and heat at 95 °C for 10 min.

[0150] C. Detection of the activities of TdT mutants in incorporating different catalytic modification substrates with different terminal starting chains

[0151] Prepare the reaction system on ice, and the specific formula is shown in Table 8 below.

[0152] Table 8

[0153] Component Concentration (total volume 25 μl) Initial strand* 1 μM <![CDATA[3’-ONH 2 -dA / T / C / GTP]]> 0.25 mM <![CDATA[CoCl 2 > 2.5 mM NaCl 100 mM Tris-HCl 7.2 50 mM TdT 0.5 mg / ml <![CDATA[H 2 O]]> Make up to 25 μl

[0154] * The initial chains include:

[0155] P1-AA: TTTTTTTTTTTTTTAA, P1-AT: TTTTTTTTTTTTTTAT,

[0156] P1-AG: TTTTTTTTTTTTTTAG, P1-AC: TTTTTTTTTTTTTTAC,

[0157] P1-TA: TTTTTTTTTTTTTTTA, P1-TT: TTTTTTTTTTTTTTT,

[0158] P1-TG: TTTTTTTTTTTTTTTTG, P1-TC: TTTTTTTTTTTTTTTC,

[0159] P1-GA: TTTTTTTTTTTTTTGA, P1-GT: TTTTTTTTTTTTTTGT,

[0160] P1-GG: TTTTTTTTTTTTTTGG, P1-GC: TTTTTTTTTTTTTTGC,

[0161] P1-CA: TTTTTTTTTTTTTTCA, P1-CT: TTTTTTTTTTTTTTCT,

[0162] P1-CG: TTTTTTTTTTTTTTCG, P1-CC: TTTTTTTTTTTTTTCC.

[0163] Reaction conditions: React at 30 °C for 30 s and heat at 95 °C for 10 min.

[0164] D: Detection by SDS-PAGE

[0165] Prepare 20% SDS-PAGE separating gel. After the gel solidifies, it can be placed in the electrophoresis tank for gel running. The specific formula of the separating gel is shown in Table 9 below.

[0166] Table 9

[0167] Component Volume (ml) <![CDATA[H 2 O]]> 0.84 30% Acr-Bis (29:1) 6.66 SDS-PAGE Separating Gel Buffer (4×) 2.5 10% APS 0.1 TEMED 0.004 Total volume 10

[0168] Take the reaction products of different TdT mutants, add 2×Loading buffer and mix well. Load 5 μl. Add 2×Loading buffer to the control group oligonucleotides and mix well. Also load 5 μl. Run the gel at 220 V for 90 min. Take pictures using a gel imager. Using 16 nt and 17 nt oligonucleotide chains of different lengths as controls, analyze the oligonucleotide extension length and the incorporation efficiency of the modified substrate, and screen TdT mutants with better incorporation effects.

[0169] 4. Result verification

[0170] (1) Detection of purified TdT mutant protein by SDS-PAGE

[0171] The results are as Figure 1 shown. The SDS-PAGE results show that the target protein can be obtained after purification and there are few impurity proteins. This method can be used to purify TdT mutants.

[0172] (2) Detection of the activity of TdT mutants in incorporating catalytic modified substrates into different terminal starting chains

[0173] The results are as Figure 2As shown, the electrophoresis results indicate that for 16 double-ended starting chains, the reaction extent of TdT 133 is better than that of TdT 4 for all of them; for the 16 double-ended starting chains, TdT113 and 117 can almost completely react, which is better than TdT4 and TdT133. TdT113 has lower activity when starting with the AA-ended chain, while TdT117 can completely react; however, TdT117 has lower activity when starting with the CC-ended chain, while TdT-113 can completely react. For the 16 double-ended starting chains, except for the CC end, TdT164 can almost completely react, with similar activity to TdT117, but both have a preference for the CC-ended starting chain.

[0174] The comprehensive results show that TdT 113 / 164 cannot completely react with the CC-ended starting chain, and the reaction extent is about 50%. It can almost completely react with the AC-ended starting chain, but there are still a small number of starting chains remaining. There is also a small amount remaining in the reaction of TdT 164 with the CG-ended starting chain.

[0175] Only the CC end of TdT228 is not completely reacted, and the reaction extent is about 50%. TdT230 / 231 cannot completely react with the CC-ended starting chain, and the reaction extent is about 50%, but they can completely react with the other 15 double-ended chains.

[0176] T4CC8 has a poor reaction with the C-ended starting chain, but can completely react with others. TdT95 can completely react with all except for the poor reaction at the CC end.

[0177] (3) Detection of the activity of TdT mutants in incorporating catalytic modification substrates with different end-starting chains (increasing metal ion concentration)

[0178] As Figure 3 shown, the electrophoresis results show that TdT 103 and TdT 104 can completely react with 7 C-containing double-ended chains. When the concentration of Co 2+ is increased to 2.5 mM, TdT228 and TdT 231 can completely react with 7 C-containing double-ended chains.

[0179] (4) Detection of the activity of TdT mutants in incorporating different catalytic modification substrates with different end-starting chains

[0180] As Figure 4 shown, for the incorporation reaction of TdT 104 and TdT 228 with four substrates for 16 different end-starting chains, TdT 104 and TdT 228 do not react completely only when dTTP is the substrate and the starting chain ends with CG. In other cases, they can completely react.

[0181] The sequence of wild-type avian TdT is as shown in SEQ ID.NO.1:

[0182] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0183] The amino acid sequence of TdT4 is shown as SEQ ID.NO.2 below:

[0184] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0185] The amino acid sequence of TdT95 is shown as SEQ ID.NO.3 below:

[0186] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGEIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0187] The amino acid sequence of TdT103 is as shown in SEQ ID.NO.4:

[0188] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGQTGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0189] The amino acid sequence of TdT104 is as shown in SEQ ID.NO.5:

[0190] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGQMGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0191] The amino acid sequence of TdT113 is shown as SEQ ID.NO.6 below:

[0192] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFTVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0193] The amino acid sequence of TdT117 is shown as SEQ ID.NO.7 below:

[0194] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYSFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0195] The amino acid sequence of TdT133 is as shown in SEQ ID.NO.8:

[0196] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHLQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0197] The amino acid sequence of TdT164 is as shown in SEQ ID.NO.9:

[0198] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSKQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0199] The amino acid sequence of TdT228 is shown as SEQ ID.NO.10 below:

[0200] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFSVMAENYSFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0201] The amino acid sequence of TdT230 is shown as SEQ ID.NO.11 below:

[0202] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFTVMAENYSFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSKQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0203] The amino acid sequence of TdT231 is shown as SEQ ID.NO.12 below:

[0204] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFSVMAENYSFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSKQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0205] The amino acid sequence of T4CC8 is shown as SEQ ID.NO.13 below:

[0206] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLTLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0207] The above-described embodiments merely represent several implementation manners of the present application, facilitating a specific and detailed understanding of the technical solutions of the present application, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A mutant of terminal deoxynucleotidyl transferase, characterized in that, the amino acid sequence of the mutant of terminal deoxynucleotidyl transferase has one or more amino acid substitutions compared with the sequence shown in SEQ ID NO: 1, and the substitution sites include at least one of positions 47, 54, 158, 210, 212, 213, 214, 273 and 332.

2. The mutant of terminal deoxynucleotidyl transferase according to claim 1, characterized in that, the amino acid substitution includes at least one of E47T, E47S, E54S, K158T, R210L, K212G, N213E, N213Q, I214T, I214M, F273L and R332K.

3. The mutant of terminal deoxynucleotidyl transferase according to claim 1 or 2, characterized in that, the amino acid substitution includes one of the following 12 combinations: Combination (1): R210L and K212G; Combination (2): R210L, K212G and N213E; Combination (3): R210L, K212G, N213Q and I214T; Combination (4): R210L, K212G, N213Q and I214M; Combination (5): R210L, K212G and E47T; Combination (6): R210L, K212G and E54S; Combination (7): R210L, K212G and F273L; Combination (8): R210L, K212G and R332K; Combination (9): R210L, K212G, E47S and E54S; Combination (10): R210L, K212G, E47T, E54S and R332K; Combination (11): R210L, K212G, E47S, E54S and R332K; Combination (12): R210L, K212G and K158T.

4. A nucleic acid molecule encoding the mutant of terminal deoxynucleotidyl transferase according to any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid molecule according to claim 4.

6. The expression vector according to claim 5, which includes a plasmid; Optionally, the plasmid includes pET-28a plasmid.

7. A host cell comprising the nucleic acid molecule according to claim 4, or comprising the expression vector according to claim 5 or 6; Optionally, the host cell includes an Escherichia coli cell; Further optionally, the Escherichia coli includes E. coli BL21(DE3).

8. A method for producing a mutant of terminal deoxynucleotidyl transferase, which comprises the following steps: culturing the host cell according to claim 7; and, isolating the mutant from the obtained culture.

9. A nucleic acid fragment synthesis kit, which includes the mutant according to claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 or 6, or the host cell according to claim 7.

10. The kit according to claim 9, further comprising other nucleic acid fragment synthesis reagents; Optionally, the other nucleic acid fragment synthesis reagents include modified dNTPs, Co 2+ , Na + , and one or more of reaction buffers; Optionally, the reaction buffer comprises 45 mM to 55 mM Tris-HCl buffer with a pH value of 7.0 to 7.4.

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