End-terminal deoxynucleotidyl transferase mutants and uses thereof

Abstract

The application belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a terminal deoxynucleotidyl transferase mutant and application thereof. The application is obtained by analyzing the structure of wild-type bird TdT, and specifically designing the amino acid sites of glutamic acid (E) at the 47th position, glutamic acid (E) at the 54th position, lysine (K) at the 158th position, arginine (R) at the 210th position, lysine (K) at the 212th position, asparagine (N) at the 213th position, isoleucine (I) at the 214th position, phenylalanine (F) at the 273th position and arginine (R) at the 332th position which affect the activity of the enzyme. The terminal deoxynucleotidyl transferase mutant obtained by the application can effectively improve the activity of the enzyme.

Description

Technical Field

[0001] This application belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a terminal deoxynucleotidyl transferase mutant and its application. Background Technology

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

[0003] With increasing oligonucleotide synthesis length, the error rate of chemical methods tends to rise, and the product yield also decreases significantly. Moreover, the synthesis process requires a large number of chemical reagents, involving strong acids and strong oxidants, and the resulting waste liquids and gases cause serious environmental pollution, leading to high subsequent treatment costs.

[0004] In recent years, experts and scholars have focused on biosynthetic methods that do not rely on chemical reagents. Bioenzymatic DNA synthesis technology is usually carried out in an aqueous environment, which can effectively avoid the above-mentioned problems and is expected to synthesize longer DNA molecules at a lower cost.

[0005] Enzymatic methods encompass biosynthesis techniques such as terminal deoxynucleotidyl transferase (TdT) catalysis, coupling methods, mixed enzyme methods, and metal ion regulation. TdT and some DNA polymerases can directly catalyze DNA strand synthesis without relying on existing DNA template molecules. Combined with in vivo assembly methods such as homologous recombination, this can improve the length and accuracy of oligonucleotide synthesis by several orders of magnitude, significantly enhancing the ability to design and construct using synthetic biology. Compared to chemical DNA synthesis, enzymatic methods hold immense promise and are expected to create significant value in terms of synthetic length and yield.

[0006] TdT is a template-independent enzyme that typically extends DNA strands randomly, adding four natural bases to the 3' end of the DNA strand. Nucleotide monomers with reversible termination groups are chemically synthesized, and then TdT enzymes are used to continuously add bases to the end of the synthesized sequence, extending only a single target base at a time. The termination group is then removed, and the synthesis of the next target base begins. This two-step process completes one round of base incorporation.

[0007] However, the activity of terminal transferase mutants obtained based on traditional techniques still cannot fully meet practical needs. Summary of the Invention

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

[0009] This application provides a mutant of terminal deoxynucleotidyl transferase, wherein 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.

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

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

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

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

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

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

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

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

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

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

[0024] This application also provides a nucleic acid molecule that encodes a mutant of the terminal deoxynucleotidyl transferase described above.

[0025] This application also provides an expression vector comprising the aforementioned nucleic acid molecule.

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

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

[0028] This application also provides a host cell that contains the nucleic acid molecule or the expression vector described herein.

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

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

[0031] This application also provides a method for producing a mutant of terminal deoxynucleotidyl transferase, comprising the following steps: culturing the host cells; and isolating the mutant from the resulting culture.

[0032] This 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 also includes other nucleic acid fragment synthesis reagents.

[0034] In one embodiment, the other nucleic acid fragment synthesis reagents include modified dNTPs, Co... 2+ Na + And one or more of the reaction buffer solution.

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

[0036] Compared to traditional technologies, the beneficial effects of this application include:

[0037] This application analyzes the structure of TdT in wild-type birds and specifically designs amino acid sites that affect the enzyme activity, namely glutamic acid at position 47 (E), glutamic acid at position 54 (E), lysine at position 158 (K), arginine at position 210 (R), lysine at position 212 (K), asparagine at position 213 (N), isoleucine at position 214 (I), phenylalanine at position 273 (F), and arginine at position 332 (R). The mutant terminal deoxynucleotidyl transferase obtained by this application can effectively enhance the enzyme activity. Attached Figure Description

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

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

[0040] Figure 2 SDS-PAGE image of the incorporation effect of 3'-ONH2-dATP into the substrate catalytically modified by TdT4 / 113 / 117 / 133 / 164 / 228 / 230 / 231 / 95 / T4CC8;

[0041] Figure 3 SDS-PAGE analysis of the incorporation of the TdT228 / 231 / 103 / 104 catalytically modified substrate 3'-ONH2-dATP;

[0042] Figure 4 SDS-PAGE analysis of four modified substrates incorporated into the TdT104 / 228 catalyst. Detailed Implementation

[0043] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. 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 modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller 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 this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] the term

[0046] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0047] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0048] As used herein, the terms "and / or," "or / and," and "and / or" encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions 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 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 the protein sequence is indicated by a note such as R210L, where R210L 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 a mutant of the reference protein).

[0050] The term "SDS-PAGE" refers to sodium dodecyl sulfate polyacrylamide gel electrophoresis, a commonly used protein expression analysis technique in polyacrylamide gel electrophoresis. This technique works by separating proteins in a sample based on their molecular weight differences within the gel. SDS-PAGE is essential in experiments involving the expression and purification of exogenous proteins in *E. coli*, typically used to detect protein expression levels (expression amount, expression distribution) and analyze the purity of the target protein.

[0051] The term "qPCR" stands for Quantitative Real-time PCR, a method that uses fluorescent chemicals to measure the total amount of product after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. It involves quantifying specific DNA sequences in a sample using internal or external controls.

[0052] Real-time PCR monitors the PCR process in real time using fluorescence signals. Because there is a linear relationship between the template's Ct value and its initial copy number during the exponential phase of PCR amplification, it serves as a basis for quantification.

[0053] The term "vector" refers to a nucleic acid molecule capable of transporting or transferring foreign nucleic acid molecules. This term encompasses both expression vectors and transcription vectors. An "expression vector" is a vector capable of expressing an insert in target cells and typically contains control sequences (such as enhancer, promoter, and terminator sequences) that drive the expression of the insert. A "transcription vector" is a vector capable of being transcribed but not translated. Transcription vectors are used to amplify their inserts. The foreign nucleic acid molecule is referred to as the "insertion" or "transgenic gene." Vectors typically consist of an insert and a larger sequence that serves as the vector's backbone. Based on their structure or origin, major types of vectors include plasmid vectors, granular vectors, phage vectors (such as λ phage), viral vectors (such as adenovirus vectors), and artificial chromosomes.

[0054] The term "host cell" refers to the cell within which a vector can reproduce and express its DNA or RNA. This cell can be prokaryotic or eukaryotic, and the recipient cell is also called the host cell. Recipient cells include prokaryotic recipient cells (most commonly E. coli), eukaryotic recipient cells (most commonly yeast), animal cells, and insect cells (which are also eukaryotic recipient cells). Among prokaryotic recipient cells, E. coli is the most commonly used host cell.

[0055] This application provides a mutant of terminal deoxynucleotidyl transferase, wherein 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.

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

[0057] In one embodiment, the amino acid substitution includes one of the following 12 combinations:

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

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

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

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

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

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

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

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

[0066] Combinations (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 that encodes a mutant of the terminal deoxynucleotidyl transferase described above.

[0071] This application also provides an expression vector comprising the aforementioned nucleic acid molecule. There are no particular limitations on the vector into which the nucleic acid encoding the mutant terminal deoxynucleotidyl transferase of this application is inserted, and any vector commonly used in the art can be used. Vectors capable of autonomous replication in host cells or vectors capable of integrating into the host chromosome can be used.

[0072] Optionally, it includes plasmids. This is understood to include plasmid vectors, phage vectors, viral vectors, etc. As plasmid vectors, plasmids suitable for the host to be used, such as plasmids derived from *Escherichia coli*, plasmids derived from *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 a phage vector, for example, λ phage can be used. As a viral vector, 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 described nucleic acid molecule or the described expression vector. There are no particular limitations on the methods used to introduce the expression vector into the host, as long as they can introduce the nucleic acid into the host, and examples include methods using calcium ions, electroporation, protoplasts, and lithium acetate.

[0075] In one 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, comprising the following steps: culturing the host cells; and isolating the mutant from the resulting culture.

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

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

[0080] Alternatively, other nucleic acid fragment synthesis reagents include modified dNTPs, Co... 2+ Na + And one or more of the reaction buffer solution.

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

[0082] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0083] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0084] Example 1

[0085] I. Experimental Materials

[0086] 1. Experimental apparatus

[0087] PCR instrument, clean bench, shaker, high-throughput non-contact ultrasonic homogenizer, centrifuge, refrigerated centrifuge, protein purification instrument, magnetic separator, electrophoresis apparatus, electrophoresis tank, gel imaging system, microwave oven.

[0088] 2. Experimental Consumables

[0089] Pipettes, disposable pipette tips, EP tubes, disposable plates, power strips, test tubes, shake flasks, centrifuge tubes, centrifuge cups, protein purification magnetic beads, protein purification columns, nickel packing materials.

[0090] 3. Preparation of experimental reagents

[0091] The formulations of the fermentation medium are shown in Table 1. The protein purification buffer includes equilibration buffer and washing buffer, as shown in Tables 2-4: Table 1

[0092] Components concentration trypsin 10g / L yeast powder 5g / L NaCl 10g / L

[0093] The formulation of the equilibration buffer is shown in Table 2:

[0094] Table 2

[0095] Components concentration Tris-HCl pH 7.2 50mM NaCl 300mM

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

[0097] Table 3

[0098] Components concentration Tris-HCl pH 7.2 50mM NaCl 300mM imidazole 50mM

[0099] The elution buffers are shown in Table 4:

[0100] Table 4

[0101] Components concentration Tris-HCl pH 7.2 50mM NaCl 300mM imidazole 300mM glycerin 10％

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

[0103] 5. Experimental medium: pET series

[0104] II. Specific Process

[0105] 1. Gene design and synthesis

[0106] To improve the catalytic activity of TdT for modified substrates, the structure of wild-type bird TdT was analyzed, and the amino acid sites affecting the enzyme activity—glutamic acid at position 47 (E), glutamic acid at position 54 (E), lysine at position 158 (K), arginine at position 210 (R), lysine at position 212 (K), asparagine at position 213 (N), isoleucine at position 214 (I), phenylalanine at position 273 (F), and arginine at position 332 (R)—were optimized.

[0107] While maintaining the secondary structure, the following mutations can be made: glutamic acid (E) at position 47 can be mutated to threonine (T) / serine (S) (E47T / E47S); glutamic acid (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 glutamic acid (E) or glutamine (Q) (N213E / N213Q); and so on. The isoleucine at position 14 (I) was mutated to threonine (T) / methionine (M) (1214T / I214M), the phenylalanine at position 273 (F) was mutated to leucine (L) (F273L), and the arginine at position 332 (R) was mutated to lysine (K) (R332K), resulting in the following 12 avian TdT mutants: TdT4, TdT95, TdT103, TdT104, TdT113, TdT117, TdT133, TdT164, TdT228, TdT230, TdT231, and T4CC8. The catalytic activity of the TdT mutants was then 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 wild-type and mutant TdT were designed, the genes were synthesized (by Nanjing Qingke Biotechnology Co., Ltd.) and the codons were optimized and ligated into the pET-28a plasmid to obtain expression vectors for the wild-type and mutant TdT.

[0112] 2. Induced expression

[0113] Expression plasmids of the TdT mutant were extracted from the top 10 E. coli cells, and 5-10 μl were slowly added to E. coli BL21(DE3) competent cells. After mixing, the cells were incubated on ice for 30 min, heat-shocked at 42℃ for 45-90 s, and then incubated on ice for 1-2 min. 900 μl of LB medium was added, and the cells were incubated at 37℃ for 220 rpm for 1 h. After centrifugation at 5000 rpm for 3 min, a small amount of supernatant was collected, mixed thoroughly, and spread onto LB agar plates containing kanamycin. The plates were incubated overnight at 37℃. Single clones were inoculated into 5 mL of LB agar containing kanamycin and incubated overnight at 37℃ for 220 rpm. The bacterial culture was then inoculated into 100 mL of LB agar containing kanamycin at a 1% inoculation rate and incubated at 37℃ for 220 rpm until the OD reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mM, and the cells were induced overnight at 16℃ for 120 rpm.

[0114] 3. Protein purification

[0115] (1) Cell disruption

[0116] After the induction of expression was completed, 40 mL of bacterial culture was centrifuged at 3500 rpm for 15 min, the supernatant was discarded, and the cells were resuspended in 1.5 mL of equilibration buffer. A final concentration of 1 mM protease inhibitor was added, and the cells were lysed using a high-throughput non-contact ultrasonic disruptor at a temperature of 4℃, a power of 100%, an interval of 3 s, and a total duration of 60 min. After disruption, the cells were centrifuged at 12000 rpm for 15 min at 4℃, and the supernatant was collected.

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

[0118] Take 150 μl of the magnetic bead suspension and place it on a magnetic separator. After the solution becomes clear, discard the supernatant with a pipette. Add 200 μl of equilibration buffer, and repeatedly pipette 5-10 times. Place it on a magnetic separator again, discard the supernatant with a pipette, and repeat the washing process twice.

[0119] (3) Magnetic beads bind to target proteins

[0120] Add the supernatant from centrifugation to the prepared magnetic beads and mix by inverting. Incubate at 4°C and 40 rpm for 1 hour, then centrifuge to remove the supernatant. Remove the centrifuge tube from the magnetic separator for washing. Add 400 μl of washing buffer to the centrifuge tube and repeatedly pipette 5-10 times to remove the supernatant. Repeat once. Add 50 μl to 100 μl of elution buffer to the centrifuge tube, incubate at 4°C and 40 rpm for 10 minutes, then centrifuge to obtain the target protein.

[0121] (4) SDS-PAGE detection

[0122] A 4%-20% SDS-PAGE precast gel (provided by Hubei Qingke Biotechnology Co., Ltd.) was placed in an electrophoresis tank for gel running. Different TdT mutant proteins were added to loading buffer and mixed well. 20 μl of the sample was loaded, and 5 μl of the marker was loaded. The gel was run at 160V for 30 min. The gel was then stained with staining solution for 15 min. After cooling, the gel was photographed using a gel imaging system. The size of the marker band was used as a control to analyze whether the size of the target protein band was correct.

[0123] (5) Protein concentration determination

[0124] After washing with ultrapure water, the sample was zeroed with elution buffer, and then the concentration of the purified target protein and the 260 / 280 value were determined using a SAM 4000.

[0125] (6) TdT activity detection

[0126] A. Activity assay of TdT mutants on substrates with different terminal initiation chains for catalytic modification.

[0127] The reaction system was prepared on ice, and the specific formulation is shown in Table 6 below:

[0128] Table 6

[0129] Components Concentration (total volume 25 μl) Initial chain* 1μM 3'-ONH2-dATP 0.25mM <![CDATA[CoCl2]]> 0.25mM NaCl 100mM Tris-HCl 7.2 50mM TdT 0.5 mg / m1 <![CDATA[H2O]]> Make up to 25 μl

[0130] *The initial chain includes:

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

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

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

[0134] P1-TG: TTTTTTTTTTTTTTTG, 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] P 1-CG: TTTTTTTTTTTTTTCG, P1-CC: TTTTTTTTTTTTTTCC.

[0139] The reaction conditions were: 30℃ for 30s, followed by heating at 95℃ for 10min.

[0140] B. Activity assay of TdT mutants on substrates with different terminal initiation chains (increased metal ion concentration)

[0141] The reaction system was prepared on ice, and the specific formulation is shown in Table 7 below:

[0142] Table 7

[0143] Components Concentration (total volume 25 μl) Initial chain* 1μM 3'-ONH2-dATP 0.25mM <![CDATA[CoCl2]]> 2.5mM NaCl 100mM Tris-HCl 7.2 50mM TdT 0.5mg / ml <![CDATA[H2O]]> Make up to 25 μl

[0144] The initial chain includes:

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

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

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

[0148] P1-CC: TTTTTTTTTTTTTTCC.

[0149] Reaction conditions: react at 30℃ for 30s, then heat at 95℃ for 10min.

[0150] C. Activity assay of TdT mutants with different catalytically modified substrates incorporated into different terminal starting chains.

[0151] The reaction system was prepared on ice, and the specific formulation is shown in Table 8 below.

[0152] Table 8

[0153] Components Concentration (total volume 25 μl) Initial chain* 1μM <![CDATA[3’-ONH2-dA / T / C / GTP]]> 0.25mM <![CDATA[CoCl2]]> 2.5mM NaCl 100mM Tris-HCl 7.2 50mM TdT 0.5mg / ml <![CDATA[H2O]]> Make up to 25 μl

[0154] *The initial chain includes:

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

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

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

[0158] P1-TG: TTTTTTTTTTTTTTTG, 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℃ for 30s, then heat at 95℃ for 10min.

[0164] D: SDS-PAGE detection

[0165] Prepare a 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] Components Volume (ml) <![CDATA[H2O]]> 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] The reaction products of different TdT mutants were added to 2× Loading buffer and mixed well, and 5 μl of each sample was loaded. The control oligonucleotides were added to 2× Loading buffer and mixed well, and 5 μl of each sample was loaded. The gel was run at 220V for 90 min, and the gel was photographed using a gel imaging system. Oligonucleotide chains of different lengths, 16nt and 17nt, were used as controls to analyze the oligonucleotide extension length and substrate incorporation efficiency, and to screen for TdT mutants with better incorporation effects.

[0169] 4. Result Verification

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

[0171] The results are as follows Figure 1 As shown in the SDS-PAGE results, the target protein can be obtained after purification with fewer impurities, and this method can be used to purify the TdT mutant.

[0172] (2) Detection of the catalytic activity of TdT mutants on substrates with different terminal initiation chains

[0173] The results are as follows Figure 2As shown, the electrophoresis results indicate that for all 16 paired-terminal initiation chains, TdT 133 showed a higher reactivity than TdT 4. For all 16 paired-terminal initiation chains, TdT 113 and 117 reacted almost completely, outperforming TdT 4 and TdT 133. TdT 113 showed lower activity when initiating with AA-terminal chains, while TdT 117 reacted completely. However, TdT 117 showed lower activity when initiating with CC-terminal chains, while TdT 113 reacted completely. For all 16 paired-terminal initiation chains except CC-terminal chains, TdT 164 reacted almost completely, showing similar activity to TdT 117, but both showed a preference for CC-terminal initiation chains.

[0174] The overall results indicate that TdT 113 / 164 does not fully react with CC-terminal initiating chains, with a reaction rate of approximately 50%. It reacts almost completely with AC-terminal initiating chains, but a small amount of initiating chains remain. TdT 164 also shows a small amount of residual reaction with CG-terminal initiating chains.

[0175] TdT228 only has incomplete reaction at the C-terminus, with a reaction rate of approximately 50%. TdT230 / 231 cannot fully react with the C-terminus initiating chain, with a reaction rate of approximately 50%, but can fully react with the other 15 dual-terminus chains.

[0176] T4CC8 reacts poorly with chains starting with a C-terminus, but reacts completely with all others. TdT95 reacts completely with all others except with a C-terminus.

[0177] (3) Detection of the catalytic activity of TdT mutants on substrates with different terminal initiation chains (increasing metal ion concentration)

[0178] like Figure 3 As shown, the electrophoresis results indicate that TdT 103 and TdT 104 react completely with all seven C-terminated compounds, while TdT 228 and TdT 231 react completely with Co. 2+ When the concentration was increased to 2.5 mM, it could completely react with all 7 C-terminal compounds.

[0179] (4) Detection of the activity of TdT mutants in incorporating different catalytically modified substrates with different terminal starting chains

[0180] like Figure 4 As shown, TdT 104 and TdT 228 underwent four substrate incorporation reactions for 16 different end-starting chains. TdT 104 and TdT 228 did not react at all when dTTP was the substrate and the CG end-starting chain was used, but they reacted completely in all other cases.

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

[0182] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

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

[0184] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

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

[0186] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGEIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

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

[0188] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGQTGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

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

[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] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRD IIEEIIIEGEESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLTLSKMQKAGLLYYEDLVSCVSKAAEADAVSLIVKNTVC TFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHGLDYVEPWERNA

[0207] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A mutant of a terminal deoxynucleotidyl transferase, characterized in that, The mutant of the terminal deoxynucleotidyl transferase is based on SEQ ID NO: 1, and the mutation is selected from any combination of the following: (1) R210L and K212G or (2) R210L, K212G and F273L.

2. A nucleic acid molecule encoding a mutant of the terminal deoxynucleotidyl transferase as described in claim 1.

3. An expression vector comprising the nucleic acid molecule of claim 2.

4. The expression vector according to claim 3, comprising a plasmid.

5. The expression vector according to claim 4, characterized in that, The plasmid includes pET-28a plasmid.

6. A host cell comprising the nucleic acid molecule of claim 2, or comprising the expression vector of any one of claims 3 to 5.

7. The host cell according to claim 6, characterized in that, The host cells include Escherichia coli cells.

8. The host cell according to claim 7, characterized in that, The Escherichia coli mentioned includes E. coli BL21(DE3).

9. A method for producing a mutant of terminal deoxynucleotidyl transferase, comprising the following steps: Culture the host cell according to any one of claims 6 to 8; and, The mutant was isolated from the resulting culture.

10. A nucleic acid fragment synthesis kit, comprising the mutant of claim 1, the nucleic acid molecule of claim 2, the expression vector of any one of claims 3 to 5, or the host cell of any one of claims 6 to 8.

11. The kit according to claim 10, further comprising other nucleic acid fragment synthesis reagents.

12. The kit according to claim 11, characterized in that, The other nucleic acid fragment synthesis reagents include modified dNTPs, Co... 2+ Na + And one or more of the reaction buffer solution.

13. The reagent kit according to claim 12, characterized in that, The reaction buffer comprises 45mM~55mM Tris-HCl buffer with a pH of 7.0~7.4.

Images