Screening method of high-activity terminal deoxynucleoside acyl transferase

Abstract

The invention relates to the technical field of gene engineering and enzyme engineering, in particular to a screening method of high-activity terminal deoxynucleoside acyl transferase. By adopting the screening method, the high-activity terminal deoxynucleoside acyl transferase can be obtained.

Description

Technical Field

[0001] The present application relates to the technical fields of genetic engineering and enzyme engineering, and in particular to a method for screening highly active terminal deoxynucleotidyl transferase. Background Art

[0002] DNA synthesis techniques mainly include chemical and biological methods. Among them, the chemical method (especially the solid-phase phosphoramidite triester synthesis method) is the most mature and widely used.

[0003] Chemical methods increase the error rate and significantly reduce product yields as the length of oligonucleotide synthesis increases. Furthermore, the synthesis process requires the use of a large number of chemical reagents, including strong acids and strong oxidants. The resulting waste liquids and gases pose a serious environmental risk, leading to high subsequent treatment costs. In recent years, experts and scholars have turned their attention to biosynthetic methods that do not rely on chemical reagents. Enzymatic DNA synthesis, typically performed in an aqueous environment, can effectively avoid these issues and promises to synthesize longer DNA molecules at a lower cost.

[0004] Enzymatic methods include terminal deoxynucleotidyltransferase (TdT)-catalyzed synthesis, coupled methods, hybrid enzyme methods, and metal ion-regulated biosynthesis techniques. TdT and some DNA polymerases can directly catalyze the synthesis of DNA chains independently of existing DNA template molecules. Combined with in vivo assembly methods such as homologous recombination, these methods can increase the length and accuracy of oligonucleotide synthesis by several orders of magnitude, significantly enhancing the ability to design and construct oligonucleotides using synthetic biology. Compared to chemical DNA synthesis, enzymatic methods hold great promise and are expected to create significant value in terms of synthesis length and yield.

[0005] TdT is a template-independent enzyme that typically extends DNA chains randomly, adding four natural bases to the 3' end of the DNA chain. Nucleotide monomers with reversible terminators are chemically synthesized, and the TdT enzyme then continuously adds bases to the ends of the synthesized fragments, extending only a single target base at a time. The terminator group is then removed, and synthesis of the next target base begins. This two-step process completes a single round of base incorporation. Effectively screening for highly active TdT is a pressing technical challenge. Summary of the Invention

[0006] Based on this, one embodiment of the present application provides a method for screening highly active terminal deoxynucleotidyl transferases. By using this screening method, highly active terminal deoxynucleotidyl transferases can be obtained.

[0007] The present invention provides a method for screening highly active terminal deoxynucleotidyl transferase, comprising the following steps:

[0008] detecting the activity of the test enzyme in catalyzing the incorporation of the modified substrate 1 into the initial chain 1 at a first reaction concentration and a first reaction time, and screening a first batch of enzymes capable of completely incorporating the modified substrate 1 from the test enzymes based on the test result 1;

[0009] detecting the activity of the first batch of enzyme in catalyzing the incorporation of the modified substrate 2 into the initial chain 2 at a second reaction concentration and a second reaction time, and screening a second batch of enzyme from the first batch of enzyme based on the detection result 2, which can completely incorporate the modified substrate 2 into the initial chain 2;

[0010] detecting the activity of the second batch of enzyme in catalyzing the incorporation of the modified substrate 3 into the initial chain 3 at a third reaction concentration and a third reaction time, and screening a third batch of enzyme from the second batch of enzyme that can completely incorporate the modified substrate 3 into the initial chain 3 based on the detection result 3;

[0011] detecting the activity of the third batch of enzyme in catalyzing the incorporation of the modified substrate 4 into the initial chain 4 at a fourth reaction concentration and a fourth reaction time, and selecting a fourth batch of enzyme from the third batch of enzyme that can completely incorporate the modified substrate 4 into the initial chain 4 as a highly active terminal deoxynucleotidyl transferase based on the detection result 4;

[0012] The second reaction time is shorter than the first reaction time;

[0013] The modified substrate 1 is the same as the modified substrate 2 but different from the modified substrate 3;

[0014] The fourth reaction concentration is lower than the first reaction concentration, the second reaction concentration and the third reaction concentration.

[0015] In some embodiments of the examples of the present application, the second reaction time includes multiple reaction time gradients, and the shortest reaction time in the multiple time gradients that can allow the modified substrate 2 to be completely incorporated into the initial chain 2 is the same as the third reaction time and the fourth reaction time.

[0016] In some embodiments of the examples of the present application, the first reaction time is 18 min-20 min, the multiple reaction time gradients in the second reaction time include 28s-32s, 0.8min-1.2min, 1.8min-2.2min, 4.8min-5.2min and 8min-12min, and the third reaction time and the fourth reaction time are 28s-32s.

[0017] In some embodiments of the examples of the present application, the modified substrate 4 is the same as the modified substrate 1 and the modified substrate 2.

[0018] In some embodiments of the examples of the present application, the modified substrate 4, the modified substrate 1 and the modified substrate 2 are all selected from any one of 3'-ONH2-dTTP, 3'-ONH2-dCTP, and 3'-ONH2-dGTP, and the modified substrate 3 is 3'-ONH2-dATP.

[0019] In some embodiments of the examples of the present application, the fourth reaction concentration includes multiple reaction concentration gradients, and the fourth batch of enzyme has a reaction concentration corresponding to a reaction concentration gradient with a lower concentration among the multiple reaction concentration gradients.

[0020] In some embodiments of the examples of the present application, the first reaction concentration, the second reaction concentration, and the third reaction concentration are each independently 0.45 mg / mL-0.55 mg / mL.

[0021] In some embodiments of the examples of the present application, the first reaction concentration, the second reaction concentration and the third reaction concentration are the same.

[0022] In some embodiments of the present invention, the screening method satisfies one or more of the following conditions:

[0023] (1) The catalytic reaction temperature is 28°C-32°C, and after the reaction is completed, the catalytic reaction system is heated at 92°C-96°C for 8min-12min;

[0024] (2) the initial strand 1, initial strand 2, initial strand 3, and initial strand 4 each independently comprise polythymidine nucleotides;

[0025] (3) In each catalytic reaction system, the corresponding initial chain concentration is 0.8 μM-1.2 μM, the CoCl2 concentration is 0.22 mM-0.26 mM, the NaCl concentration is 95 mM-105 mM, and the buffer is 48 mM-52 mM Tris-HCl buffer at pH 7.1-7.3;

[0026] (4) The enzyme to be tested includes multiple wild-type terminal deoxynucleotidyl transferase and its mutants; and

[0027] (5) Detection methods include polyacrylamide gel electrophoresis.

[0028] In some embodiments of the examples of the present application, the enzyme to be tested includes multiple terminal deoxynucleotidyl transferases with sequences shown in SEQ ID NO.1 to SEQ ID NO.7.

[0029] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0031] Figure 1 This is the TdT3,4SDS-PAGE detection image;

[0032] Figure 2 These are the images of TdT23, 39, 45, 91, and 131 SDS-PAGE detection;

[0033] Figure 3 This is the SDS-PAGE detection image of TdT112, 114, and 116;

[0034] Figure 4 Figure 118, 119, and 120 are SDS-PAGE detection images of TdT;

[0035] Figure 5 This is the SDS-PAGE detection image of TdT121, 122, and 130;

[0036] Figure 6 This is the SDS-PAGE detection diagram of the incorporation of the modified substrate 3'-ONH2-dCTP catalyzed by TdT3 and 4;

[0037] Figure 7 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT23, 39, 45, 91, and 131;

[0038] Figure 8 The figure shows the SDS-PAGE detection of the incorporation of the modified substrate 3'-ONH2-dCTP catalyzed by TdT 119, 121, and 122;

[0039] Figure 9 The SDS-PAGE detection diagram shows the incorporation of the modified substrate 3'-ONH2-dCTP catalyzed by TdT112, 114, 116, 118, 120, and 130;

[0040] Figure 10 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT4, 45, 91, and 131;

[0041] Figure 11 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT4, 45, 91, and 131;

[0042] Figure 12 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT4, 45, 91, and 131;

[0043] Figure 13 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT4, 45, 91, and 131;

[0044] Figure 14 The SDS-PAGE detection diagram shows the incorporation of the modified substrate 3'-ONH2-dATP catalyzed by TdT4, 45, 91, and 131;

[0045] Figure 15 The SDS-PAGE detection diagram shows the incorporation of the modified substrate 3'-ONH2-dATP catalyzed by TdT4, 91, and 131;

[0046] Figure 16 The figure shows the SDS-PAGE detection of the incorporation of the modified substrate 3'-ONH2-dATP catalyzed by TdT4 and 131;

[0047] Figure 17 This is the SDS-PAGE detection diagram of the incorporation of the modified substrate 3'-ONH2-dATP catalyzed by low concentrations of TdT4 and 131;

[0048] Figure 18 To add Mg 2+ The incorporation of the modified substrate 3'-ONH2-dA / T / C / GTP catalyzed by TdT131 was detected by SDS-PAGE;

[0049] Figure 19 To add Zn 2+ and Mg 2+ After or add Co 2+ 、Zn 2+ and Mg 2+ The incorporation of TdT131-catalyzed modified substrate 3'-ONH2-dA / T / C / GTP into SDS-PAGE was detected. DETAILED DESCRIPTION

[0050] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0052] the term

[0053] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0054] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by 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 technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0055] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0056] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0057] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0058] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0059] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0060] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0061] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0062] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0063] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0064] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0065] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0066] The present invention provides a method for screening highly active terminal deoxynucleotidyl transferase, comprising the following steps:

[0067] detecting the activity of the test enzyme in catalyzing the incorporation of the modified substrate 1 into the initial chain 1 at a first reaction concentration and a first reaction time, and screening a first batch of enzymes capable of completely incorporating the modified substrate 1 from the test enzymes based on the test result 1;

[0068] detecting the activity of the first batch of enzyme in catalyzing the incorporation of the modified substrate 2 into the initial chain 2 at a second reaction concentration and a second reaction time, and screening a second batch of enzyme from the first batch of enzyme based on the detection result 2, which can completely incorporate the modified substrate 2 into the initial chain 2;

[0069] detecting the activity of the second batch of enzyme in catalyzing the incorporation of the modified substrate 3 into the initial chain 3 at a third reaction concentration and a third reaction time, and screening a third batch of enzyme from the second batch of enzyme that can completely incorporate the modified substrate 3 into the initial chain 3 based on the detection result 3;

[0070] detecting the activity of the third batch of enzyme in catalyzing the incorporation of the modified substrate 4 into the initial chain 4 at a fourth reaction concentration and a fourth reaction time, and selecting a fourth batch of enzyme from the third batch of enzyme that can completely incorporate the modified substrate 4 into the initial chain 4 as a highly active terminal deoxynucleotidyl transferase based on the detection result 4;

[0071] The second reaction time is shorter than the first reaction time;

[0072] The modified substrate 1 is the same as the modified substrate 2 but different from the modified substrate 3;

[0073] The fourth reaction concentration is lower than the first reaction concentration, the second reaction concentration and the third reaction concentration.

[0074] In some examples, the second reaction time includes multiple reaction time gradients, and the shortest reaction time in the multiple time gradients that allows the modified substrate 2 to be completely incorporated into the initial chain 2 is the same as the third reaction time and the fourth reaction time.

[0075] Optionally, the first reaction time is 18 min-20 min (for example, 18, 18.5, 19, 19.5, 20 min), and the multiple reaction time gradients in the second reaction time include 28 s-32 s (for example, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32 s), 0.8 min-1.2 min (for example, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2 min), 1.8 min-2.2 min (for example, 1.8, 1.85, 1.15, 1.2 min), and 2.5 min-3.5 min (for example, 3.5 ... The third reaction time and the fourth reaction time are 28s-32s (for example, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32s).

[0076] In some examples, the modified substrate 4 is the same as the modified substrate 1 and the modified substrate 2.

[0077] In some examples, the modified substrate 4, the modified substrate 1, and the modified substrate 2 are all selected from any one of 3'-ONH2-dTTP, 3'-ONH2-dCTP, and 3'-ONH2-dGTP, and the modified substrate 3 is 3'-ONH2-dATP.

[0078] In some examples, the fourth reaction concentration includes a plurality of reaction concentration gradients, and the fourth batch of enzyme has a reaction concentration corresponding to a reaction concentration gradient with a lower concentration among the plurality of reaction concentration gradients.

[0079] In some examples, the first reaction concentration, the second reaction concentration, and the third reaction concentration are each independently 0.45 mg / mL-0.55 mg / mL.

[0080] In some examples, the first reaction concentration, the second reaction concentration, and the third reaction concentration are the same.

[0081] In some examples, the screening method satisfies one or more of the following conditions:

[0082] (1) The catalytic reaction temperature is 28° C.-32° C. (e.g., 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32° C.), and after the reaction, the catalytic reaction system is heated at 92° C.-96° C. (e.g., 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96° C.) for 8 min-12 min (e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 min);

[0083] (2) the initial strand 1, initial strand 2, initial strand 3, and initial strand 4 each independently comprise polythymidine nucleotides;

[0084] (3) In each catalytic reaction system, the concentration of the corresponding initial chain is 0.8 μM-1.2 μM (for example, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2 μM), the concentration of CoCl2 is 0.22 mM-0.26 mM (for example, 0.22, 0.23, 0.24, 0.25, 0.26 mM), and the concentration of Na The concentration of Cl is 95 mM-105 mM (e.g., 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 mM), and the buffer is 48 mM-52 mM (e.g., 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52 mM) Tris-HCl buffer at pH 7.1-7.3;

[0085] (4) The enzyme to be tested includes multiple wild-type terminal deoxynucleotidyl transferase and its mutants; and

[0086] (5) Detection methods include polyacrylamide gel electrophoresis.

[0087] In some examples, the enzyme to be tested includes multiple terminal deoxynucleotidyl transferases with sequences shown in SEQ ID NO. 1 to SEQ ID NO. 7.

[0088] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0089] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0090] Example 1

[0091] 1. Methods

[0092] 1.1 Gene design and synthesis

[0093] 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 affecting the enzyme activity, arginine (R) at position 210, lysine (K) at position 212, asparagine (N) at position 213, isoleucine (I) at position 214, and methionine (M) at position 270, were optimized.

[0094] While keeping the secondary structure unchanged, the 47th glutamic acid (E) was mutated to lysine (K), glutamine (D), the 54th glutamic acid (E) was mutated to glycine (G), aspartic acid (Q), glutamine (D), leucine (L), the 73rd lysine (K) was mutated to arginine (R), valine (V), the 210th arginine (R) was mutated to leucine (L), the 212th lysine (K) was mutated to glycine (G), the 213th asparagine (N) was mutated to aspartic acid (D), the 214th isoleucine (I) was mutated to threonine (T), and the 270th methionine (M) was mutated to phenylalanine (F), resulting in the following mutants of avian wild-type TdT: TdT4, TdT91, TdT112, TdT114, TdT116, TdT118, TdT119. 119, TdT120, TdT 121, TdT 122, TdT130, and TdT 131. Meanwhile, TdT 23 (grey wolf), TdT 39 (mouse), and TdT 45 (spotted gar) from different species were used as controls to determine the catalytic activity of TdT.

[0095] After the TdT wild type and mutants were designed, the genes were synthesized (by Nanjing Qingke Biotechnology Co., Ltd.), codon optimized, and ligated into the pET-28a plasmid to obtain the expression vectors of the TdT wild type and mutants.

[0096] Table 1

[0097]

[0098] 1.2 Inducible expression

[0099] Extract the wild-type and mutant TdT expression plasmids from E. coli top10 and slowly add 5-10 μL of each to competent E. coli BL21(DE3) cells. Mix thoroughly, incubate on ice for 30 minutes, heat shock at 42°C for 45-90 seconds, and then incubate on ice for 1-2 minutes. Add 900 μL of LB and incubate at 37°C at 220 rpm for 1 hour. Centrifuge at 5000 rpm for 3 minutes. Retain a small amount of supernatant and mix thoroughly by pipetting. Plate the plate onto a kanamycin-resistant LB plate and incubate inverted at 37°C overnight. Select a single colony and inoculate it into 5 mL of kanamycin-resistant LB plate. Incubate at 220 rpm at 37°C overnight. Inoculate the culture at 1% of the inoculum into 100 mL of kanamycin-resistant LB plate. Cultivate at 37°C at 220 rpm to an OD of 0.6-0.8. Add IPTG to a final concentration of 0.5 mM and induce overnight at 16°C at 120 rpm.

[0100] 1.3 Protein purification

[0101] 1.3.1 Cell disruption

[0102] After induction of expression, 40 mL of bacterial solution was centrifuged at 3500 rpm for 15 min, the supernatant was discarded, and the cell suspension was resuspended with 1.5 mL of equilibration buffer. Protease inhibitors were added at a final concentration of 1 mM, and the cells were lysed using a high-throughput non-contact ultrasonic disruptor. The disruption temperature was 4°C, the power was 100%, the interval time was 3 s, and the total duration was 60 min. After disruption, the cell suspension was centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected.

[0103] 1.3.2 Magnetic Bead Preparation and Equilibration

[0104] Take 150 μL of the magnetic bead suspension and place it on a magnetic separator. Once the solution becomes clear, aspirate and discard the supernatant with a pipette. Add 200 μL of equilibration buffer, pipette and resuspend 5-10 times, place on a magnetic separator, aspirate and discard the supernatant with a pipette, and repeat the wash process two more times.

[0105] 1.3.3 Magnetic beads binding target protein

[0106] Add the supernatant after centrifugation to the treated magnetic beads and mix thoroughly by inverting. Invert at 40 rpm at 4°C for 1 hour, then centrifuge to remove the supernatant. Remove the centrifuge tube from the magnetic separator and wash the impurities. Add 400 μL of wash buffer to the centrifuge tube and pipette repeatedly 5-10 times, remove the supernatant, and repeat once. Add 50-100 μL of elution buffer to the centrifuge tube, invert at 40 rpm at 4°C for 10 minutes, and then centrifuge to obtain the target protein.

[0107] 1.4 SDS-PAGE detection

[0108] Use 4%-20% SDS-PAGE precast gel (provided by Hubei Qingke Biotechnology Co., Ltd.) in an electrophoresis tank for gel running. Take different TdT mutant proteins and add loading buffer and mix well. Load 20 μL of sample and 5 μL of marker. Run the gel at 160V for 30 minutes. Heat and stain the gel with staining solution for 15 minutes. After cooling, use a gel imager to take pictures. Use the marker band size as a reference to analyze whether the target protein band size is correct.

[0109] 1.5 Protein concentration determination

[0110] After washing with ultrapure water, the plate was zeroed with elution buffer, and the concentration and 260 / 280 value of the purified target protein were measured using SAM 4000.

[0111] 1.6 TdT activity detection

[0112] 1.6.1 Detection of TdT wild-type and mutant catalytic modified substrate incorporation activity

[0113] The reaction system was prepared on ice. The specific formula is shown in the following table.

[0114] Table 2

[0115] Components Concentration (total volume 50 μL) Initial Chain 1 μM <![CDATA[3’-ONH2-dCTP]]> 0.25mM <![CDATA[CoCl2]]> 0.25mM NaCl 100mM Tris-HCl 7.2 50mM TdT 0.5 mg / mL <![CDATA[H2O]]> Make up to 50 μL

[0116] Reaction conditions: 30°C for 20 min, heating at 95°C for 10 min.

[0117] 1.6.2 Shortening the catalytic time to detect the activity of TdT mutants incorporating modified substrates

[0118] The reaction system was prepared on ice. The specific formula is shown in the following table.

[0119] Table 3

[0120] Components Concentration (total volume 50 μL) Initial Chain 1 μM <![CDATA[3’-ONH2-dCTP]]> 0.25mM <![CDATA[CoCl2]]> 0.25mM NaCl 100mM Tris-HCl 7.2 50mM TdT 0.5 mg / mL <![CDATA[H2O]]> Make up to 50 μL

[0121] Reaction conditions: 30°C for 30s / 1min / 2min / 5min / 10min, heating at 95°C for 10min.

[0122] 1.6.3 Testing the catalytic activity of TdT mutants by replacing modified substrates

[0123] The reaction system was prepared on ice. The specific formula is shown in the following table.

[0124] Table 4

[0125]

[0126]

[0127] Reaction conditions: 30°C for 30 seconds, heating at 95°C for 10 minutes.

[0128] 1.6.4 Lowering TdT concentration to test the activity of TdT mutants incorporating modified substrates

[0129] The reaction system was prepared on ice. The specific formula is shown in the following table.

[0130] Table 5

[0131] Components Concentration (total volume 50 μL) Initial Chain 1 μM <![CDATA[3’-ONH2-dCTP]]> 0.25mM <![CDATA[CoCl2]]> 0.25mM NaCl 100mM Tris-HCl 7.2 50mM TdT 0.5 / 0.25 / 0.13 / 0.06 / 0.03mg / mL <![CDATA[H2O]]> Make up to 50 μL

[0132] Reaction conditions: 30°C for 30 seconds, heating at 95°C for 10 minutes.

[0133] 1.6.5 Effect of metal ion addition on the activity of TdT mutants in catalytically modifying substrates

[0134] The reaction system was prepared on ice. The specific formula is shown in the following table.

[0135] Table 6

[0136] Components Concentration (total volume 50 μl) <![CDATA[Initial chain *1 > 1 μM <![CDATA[3’-ONH2-dA / T / C / GTP]]> 0.25mM <![CDATA[Metal ion *2 > x NaCl 100mM Tris-HCl 7.2 50mM TdT 0.5 / 0.25 / 0.13 / 0.06 / 0.03mg / ml <![CDATA[H2O]]> Make up to 50 μl

[0137] Note:

[0138] 1. The types of initial chain additions are different. AA means that the initial chain ends with A and the modified substrate 3'-ONH2-dATP is incorporated. AT means that the initial chain ends with A and the modified substrate 3'-ONH2-dTTP is incorporated. And so on. GG means that the initial chain ends with G and the modified substrate 3'-ONH2-dGTP is incorporated.

[0139] 2. There are three types of metal ion addition: 0.25mM Co 2+ , 0.25mM Co 2+ + 2 mM Mg 2+ 、0.25mM Zn 2+ and 2 mM Mg 2+ , 0.25mM Co 2+ +0.25mM Zn 2++2mM Mg 2+ .

[0140] 1.6.6 SDS-PAGE detection

[0141] Prepare 20% SDS-PAGE separation gel and place it in the electrophoresis tank for running after the gel solidifies. The specific formula of the separation gel is shown in the table below.

[0142] Table 7

[0143] 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

[0144] The reaction products of different TdT mutants were added to 2×Loading buffer and mixed, and 5 μL was loaded. The control oligonucleotide was added to 2×Loading buffer and mixed, and 5 μL was loaded. The gel was run at 220V for 90 min, and photos were taken using a gel imager. Oligonucleotide chains of different lengths, such as 20nt and 21nt, were used as controls to analyze the oligonucleotide extension length and the modified substrate incorporation efficiency, and TdT mutants with better incorporation effects were screened.

[0145] 2. Experimental Results

[0146] 2.1 SDS-PAGE detection of purified TdT mutant protein

[0147] See the results Figures 1 to 5 .

[0148] Figure 1 This is the TdT3,4SDS-PAGE detection image;

[0149] Figure 2 These are the images of TdT23, 39, 45, 91, and 131 SDS-PAGE detection;

[0150] Figure 3 This is the SDS-PAGE detection image of TdT112, 114, and 116;

[0151] Figure 4 This is the SDS-PAGE detection image of TdT118, 119, and 120;

[0152] Figure 5 This is the SDS-PAGE detection diagram of TdT121, 122, and 130.

[0153] SDS-PAGE results showed that the target protein could be obtained after purification, and the target protein accounted for a high proportion. This method can be used to purify TdT wild type and its mutants.

[0154] 2.2 Detection of TdT wild-type and mutant catalytic modified substrate incorporation activity

[0155] See the results Figures 6 to 9 .

[0156] Figure 6 The SDS-PAGE detection diagram of TdT3 and 4 catalyzed modification of substrate 3'-ONH2-dCTP incorporation;

[0157] Figure 7 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT23, 39, 45, 91, and 131;

[0158] Figure 8 SDS-PAGE detection of TdT 119, 121, and 122-catalyzed substrate 3'-ONH2-dCTP incorporation

[0159] Figure 9 SDS-PAGE detection of the incorporation of the modified substrate 3'-ONH2-dCTP catalyzed by TdT112, 114, 116, 118, 120, and 130.

[0160] The electrophoresis results showed that TdT3, 4, 45, 91, and 131 were able to incorporate the modified substrate 3'-ONH2-dCTP within a reaction time of 20 min, but TdT23, 39, 114, 119, and 121 did not react completely, and some initial chains remained. TdT112, 118, 120, 122, and 130 did not react, as can be seen in the figure.

[0161] It can be seen that TdT3, 4, 45, 91, and 131 have high enzyme activities and can completely react the substrate. Subsequently, TdT3, 4, 45, 91, and 131 were selected for experiments to shorten the reaction time and screened for highly active mutants.

[0162] 2.3 Shortening the catalytic time to detect the activity of TdT mutants incorporating modified substrates

[0163] See the results Figures 10 to 13 .

[0164] Figure 10 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT4, 45, 91, and 131;

[0165] Figure 11 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT4, 45, 91, and 131;

[0166] Figure 12 The SDS-PAGE detection diagram shows the incorporation of 3'-ONH2-dCTP into the modified substrate catalyzed by TdT4, 45, 91, and 131;

[0167] Figure 13 This is the SDS-PAGE detection diagram of the incorporation of the modified substrate 3'-ONH2-dCTP catalyzed by TdT4, 45, 91, and 131.

[0168] The electrophoresis results showed that the enzyme activities of TdT45, 91, 131 and TdT4 were equivalent at 10 minutes and 5 minutes of reaction, and they were able to completely react with the modified substrate. Subsequently, when the reaction time was further shortened to 2 minutes and 1 minute, there was still no observable difference in enzyme activity between TdT45, 91, 131 and TdT4, and all four enzymes were able to complete all initial linked reactions.

[0169] Since TdT has low incorporation activity towards the modified substrate 3'-ONH2-dATP, the incorporation effect of the mutant on 3'-ONH2-dATP was subsequently tested.

[0170] 2.4 Detection of the catalytic activity of TdT mutants by replacing and incorporating modified substrates

[0171] See the results Figures 14 to 16 .

[0172] Figure 14 The SDS-PAGE detection diagram shows the incorporation of the modified substrate 3'-ONH2-dATP catalyzed by TdT4, 45, 91, and 131;

[0173] Figure 15 The SDS-PAGE detection diagram shows the incorporation of the modified substrate 3'-ONH2-dATP catalyzed by TdT4, 91, and 131;

[0174] Figure 16 This is the SDS-PAGE detection diagram of the incorporation of the modified substrate 3'-ONH2-dATP catalyzed by TdT4 and 131.

[0175] When 3'-ONH2-dATP was used as the modified substrate, when the reaction time was shortened to 10 minutes, TdT45 could hardly catalyze its incorporation; when the time was shortened to 1 minute, TdT4 and 91 did not completely catalyze the incorporation of the modified substrate into the initial chain, and the figure shows that there was still a remainder of the initial chain, while TdT131 was still able to react completely; when the time was shortened to 30 seconds, TdT4 still did not completely catalyze the incorporation of the modified substrate into the initial chain, and the figure shows that there was still a remainder of the initial chain, while TdT131 was still able to react completely. Therefore, it can be judged that the enzyme activity of the mutant TdT131 is better than that of the wild type and other mutants.

[0176] 2.5 Lowering TdT concentration to detect TdT mutant catalytic modified substrate incorporation activity

[0177] See the results Figure 17 .

[0178] Figure 17This is the SDS-PAGE detection diagram of the incorporation of the modified substrate 3'-ONH2-dATP catalyzed by low concentrations of TdT4 and 131.

[0179] Electrophoresis results showed that the amount of uncatalyzed initial chains remaining in TdT4 increased with increasing dilution. Unreacted initial chain bands became apparent at a 4-fold dilution of TdT4, whereas unreacted initial chain bands were only observed at a 16-fold dilution of TdT131. In summary, when equal amounts of enzyme were added to the reaction system, TdT131 reacted more completely, indicating higher enzyme activity.

[0180] 2.6 Effect of metal ion addition on the catalytic activity of TdT mutants in modifying substrates

[0181] See the results Figure 18 and Figure 19 .

[0182] Figure 17 To add Mg 2+ The results showed that compared with the above figure, only adding Co 2+ Mg was added to the reaction system 2+ There was no significant improvement.

[0183] Figure 18 To add Zn 2+ and Mg 2+ After or add Co 2+ 、Zn 2+ and Mg 2+ The SDS-PAGE detection diagram of TdT131-catalyzed modification of substrate 3'-ONH2-dA / T / C / GTP. The results showed that changing the metal ion in the reaction system to Zn 2+ +Mg 2+ After, or in Co 2+ Adding additional metal ions Zn 2+ and Mg 2+ After addition of 1% TdT 131, the incorporation activity of TdT 131 into the C-terminal initiator chain could not be improved.

[0184] Table 8

[0185]

[0186]

[0187] 3. Analysis and Discussion

[0188] In the initial screening reaction of TdT and its mutants, TdT3, 4, 23, 45, 91, and 131 could all catalyze the incorporation of the modified substrate 3'-ONH2-dATP into the initial chain. SDS-PAGE results showed that TdT23, 39, 114, 119, and 121 could not catalyze the complete incorporation of the modified substrate, and some initial chains remained. TdT112, 118, 120, 122, and 130 did not react.

[0189] When the reaction time was shortened to 2 min and 1 min, there was still no observable difference in enzyme activity between TdT45, 91, 131 and TdT4, and all four enzymes were able to complete all initial chain reactions.

[0190] When 3'-ONH2-dATP was used as the modification substrate, when the reaction time was shortened to 10 min, TdT45 could hardly catalyze its incorporation into the initial chain; when the time was shortened to 1 min, TdT4 and 91 could not completely catalyze the incorporation of the modified substrate into the initial chain; when the time was shortened to 30 s, TdT4 still failed to completely catalyze the incorporation of the modified substrate into the initial chain, while TdT131 was still able to complete the reaction.

[0191] With increasing dilution of TdT4, the amount of unreacted initial chains increased. However, with TdT131, bands of unreacted initial chains were only observed at a 16-fold dilution. In summary, when equal amounts of enzyme were added to the reaction system, TdT131 catalyzed the more complete incorporation of modified substrates into the initial chains, indicating that TdT131 has higher enzyme activity.

[0192] In summary, TdT131 exhibits high modified substrate incorporation activity, especially the reaction of modified substrates that are more difficult to incorporate can be basically catalyzed to completion, which is significantly improved compared with the wild type and other mutants.

[0193] TdT 3 (wild type, SEQ ID NO. 1):

[0194] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEIIEEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEIFAHLGLDYVEPWERNA.

[0195] TdT 4(SEQ ID NO.2):

[0196] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEIIEEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEIFAHLGLDYVEPWERNA.

[0197] TdT 91(SEQ ID NO.3):

[0198] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEIIEEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGDTGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEIFAHLGLDYVEPWERNA.

[0199] TdT 131(SEQ ID NO.4):

[0200] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEIIEEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAFDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEIFAHLGLDYVEPWERNA.

[0201] TdT 23(SEQ ID NO.5):

[0202] MDYTASPNPELQKTLPVAVKKISQYACQRRTTLNNYNNVFTDAFEVLAENYEFRENEVFSLTFQRAASVLKSLPFTIISMKDTEGIPCLGDQVKCIIEEIIEDGESSEVKAVLNDERYQSFKLFTSVFGVGLKTSEKWFRMGFRTLSKIKSDKSLKFTPMQKAGFLYYEDLVSGVTRAEAEAVGVLVKEAVGAFLPDAFVTMTGGFRNGKKMGHDVDFLITSPGSTDEDEEQLLPKVINLWERKGLLLYCDLVESTFEKLKLPSRKVDALDHFQKCFLILKLHHQRVDGGKCSQQEGKTWKAIRVDLVMCPYERRAFALLGWTGSPQFERDLRRYASHERKMILDNHALYDKTKKIFLKAESEEEIFAHLGLDYIEPWERNA.

[0203] TdT 39(SEQ ID NO.6):

[0204] MRGSGMASMTGGQQMGRDLYDDDDKDRWGSELEKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSCLAFMRASSVLKSLPFPITSMKDTEGIPCLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYCDILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMCPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA.

[0205] TdT 45(SEQ ID NO.7):

[0206] MLHIPIFPPIKKRQKLPESRNSCKYEVKFSEVAIFLVERKMGSSRRKFLTNLARSKGFRIEDVLSDAVTHVVAEDNSADELWQWLQNSSLGDLSKIEVLDISWFTECMGAGKPVQVEARHCLVKSCPVIDQYLEPSTVETVSQYACQRRTTMENHNQIFTDAFAILAENAEFNESEGPCLAFMRAASLLKSLPHAISSSKDLEGLPCLGDQTKAVIEDILEYGQCSKVQDVLCDDRYQTIKLFTSVFGVGLKTAEKWYRKGFHSLEEVQADNAIHFTKMQKAGFLYYDDISAAVCKAEAQAIGQIVEETVRLIAPDAIVTLTGGFRRGKECGHDVDFLITTPEMGKEVWLLNRLINRLQNQGILLYYDIVESTFDKTRLPCRKFEAMDHFQKCFAIIKLKKELAAGRVQKDWKAIRVDFVAPPVDNFAFALLGWTGSRQFERDLRRFARHERKMLLDNHALYDKTKKIFLPAKTEEDIFAHLGLDYIDPWQRNA。

[0207] SEQ ID NO.8 (TdT 112, (ZaTdT truncated - R210L + K212G)E47K):

[0208] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFKVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA。

[0209] SEQ ID NO.9 (TdT 114, (ZaTdT truncated - R210L + K212G)E47D):

[0210] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFDVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0211] SEQ ID NO.10 (TdT 116, (ZaTdT truncated - R210L + K212G)E54G):

[0212] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYGFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0213] SEQ ID NO.11 (TdT 118, (ZaTdT truncated - R210L + K212G)E54Q):

[0214] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYQFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0215] SEQ ID NO.12 (TdT 119, (ZaTdT truncated - R210L + K212G)E54D):

[0216] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYDFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0217] SEQ ID NO.13 (TdT 120, (ZaTdT truncated - R210L + K212G)E54L):

[0218] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYLFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0219] SEQ ID NO.14 (TdT 121, (ZaTdT truncated - R210L + K212G)K73R):

[0220] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLRSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0221] SEQ ID NO.15 (TdT 122, (ZaTdT truncated - R210L + K212G)K73V):

[0222] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLVSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0223] SEQ ID NO.16 (TdT130, (ZaTdT truncated - R210L + K212G)M270L):

[0224] MEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRLGGNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDALDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA.

[0225] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. To make the description concise, the technical features of the above-mentioned embodiments and examples are not described in detail.

[0226] All possible combinations of features are described; however, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0227] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content 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 scope of protection 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 on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. Screening method for highly active terminal deoxynucleotidyl transferase, It is characterized in that The screening method comprises the following steps: Detecting the activity of the enzyme to be tested in catalyzing the incorporation of the modified substrate 1 into the initial chain 1 at a first reaction concentration and a first reaction time, and screening a first batch of enzymes that can completely incorporate the modified substrate 1 from the enzyme to be tested according to the detection result 1; detecting the activity of the first batch of enzymes in catalyzing the incorporation of the modified substrate 2 into the initial chain 2 at a second reaction concentration and a second reaction time, and selecting a second batch of enzymes capable of completely incorporating the modified substrate 2 into the initial chain 2 from the first batch of enzymes according to the detection result 2; detecting the activity of the second batch of enzymes in catalyzing the incorporation of the modified substrate 3 into the initial chain 3 at a third reaction concentration and a third reaction time, and selecting a third batch of enzymes that can completely incorporate the modified substrate 3 into the initial chain 3 from the second batch of enzymes according to the detection result 3; detecting the activity of the third batch of enzymes in catalyzing the incorporation of the modified substrate 4 into the initial chain 4 at a fourth reaction concentration and a fourth reaction time, and selecting a fourth batch of enzymes that can completely incorporate the modified substrate 4 into the initial chain 4 from the third batch of enzymes as a highly active terminal deoxynucleotidyl transferase according to the detection result 4; The second reaction time is shorter than the first reaction time; The modified substrate 1 is the same as the modified substrate 2 but different from the modified substrate 3; The fourth reaction concentration is lower than the first reaction concentration, the second reaction concentration and the third reaction concentration.

2. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, It is characterized in that The second reaction time includes a plurality of reaction time gradients, and the shortest reaction time in the plurality of time gradients that allows the modified substrate 2 to be completely incorporated into the initial chain 2 is the same as the third reaction time and the fourth reaction time.

3. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 2, It is characterized in that The first reaction time is 18 min-20 min, the multiple reaction time gradients in the second reaction time include 28s-32s, 0.8min-1.2min, 1.8min-2.2min, 4.8min-5.2min and 8min-12min, and the third reaction time and the fourth reaction time are 28s-32s.

4. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, It is characterized in that The modified substrate 4 is the same as the modified substrate 1 and the modified substrate 2.

5. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 4, It is characterized in that The modified substrate 4, the modified substrate 1 and the modified substrate 2 are all selected from 3'-ONH 2 -dTTP, 3'-ONH 2 -dCTP, and 3'-ONH 2 -dGTP, the modified substrate 3 is 3'-ONH 2 -dATP.

6. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, It is characterized in that The fourth reaction concentration includes a plurality of reaction concentration gradients, and the fourth batch of enzymes has a reaction concentration corresponding to a reaction concentration gradient with a lower concentration among the plurality of reaction concentration gradients.

7. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 6, It is characterized in that The first reaction concentration, the second reaction concentration and the third reaction concentration are each independently 0.45 mg / mL-0.55 mg / mL.

8. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 7, It is characterized in that The first reaction concentration, the second reaction concentration and the third reaction concentration are the same.

9. The method for screening a highly active terminal deoxynucleotidyl transferase according to any one of claims 1 to 8, It is characterized in that The screening method meets one or more of the following conditions: (1) The catalytic reaction temperature is 28°C-32°C, and after the reaction, the catalytic reaction system is heated at 92°C-96°C for 8min-12min; (2) the initial strand 1, initial strand 2, initial strand 3 and initial strand 4 each independently comprise polythymine nucleotides; (3) In each catalytic reaction system, the corresponding initial chain concentration is 0.8μM-1.2μM, CoCl 2 The concentration of is 0.22mM-0.26mM, the concentration of NaCl is 95mM-105mM, and the buffer is 48mM-52mM Tris-HCl buffer at pH7.1-7.3; (4) the enzyme to be tested includes multiple wild-type terminal deoxynucleotidyl transferase and its mutants; and (5) Detection methods include polyacrylamide gel electrophoresis.

10. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 9, It is characterized in that The enzyme to be tested includes multiple terminal deoxynucleotidyl transferases whose sequences are shown in SEQ ID NO.1 to SEQ ID NO.7.

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