Screening method of high-activity terminal deoxynucleotidyl transferase

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a screening method of high-activity terminal deoxynucleotidyl transferase. The method comprises the following steps: (1) carrying out blast in a protein database by using known terminal deoxynucleotidyl transferase as a probe, and selecting a homologous enzyme which is 20% or more in sequence consistency with the probe or is similar to the probe in protein structure as a candidate enzyme; (2) classifying candidate enzymes according to species sources, screening out a plurality of candidate enzymes from each category, wherein the sequence consistency between the candidate enzymes and the probe is more than 20%, and carrying out enzyme activity analysis on the candidate enzymes; (3) carrying out researches on the optimum temperature, pH, metal ions, temperature tolerance and the like on the candidate enzyme with the best expression, and determining specific conditions most beneficial to catalytic reaction of the enzyme; and the extension and transformation conditions of the candidate enzyme on different substrates are researched. Compared with the prior art, the method has the advantages that the range of the screened TdT is wider, and the comprehensive performance of the screened enzyme is better.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for screening highly active terminal deoxynucleotidyl transferase. Background Art

[0002] Enzymatic DNA synthesis primarily relies on the enzyme terminal deoxynucleotidyltransferase (TdT). Thousands of TdT enzymes are currently available in enzyme databases, and researchers have optimized and modified TdT enzymes to improve their temperature tolerance. However, comprehensive performance analysis of different TdT enzymes is rare, which is a crucial foundation for identifying excellent starting enzymes and engineering them into better-performing mutants.

[0003] Screening for highly active terminal deoxynucleotidyl transferase (TdT) enzymes is a key challenge in enzyme engineering and molecular biology research. Commonly used screening methods include radiolabeling, fluorescence detection, microfluidic droplet sorting, phage display, FRET probes, and nanopore single-molecule detection. However, these techniques all have limitations. For example, radiolabeling, while highly sensitive, carries safety concerns; fluorescence detection is susceptible to interference from modified nucleotides; microfluidic sorting equipment is expensive and carries the risk of cross-contamination; phage display can cause conformational changes in the enzyme; FRET probes are significantly affected by salt concentration; and nanopore detection has low throughput and difficulty distinguishing enzymatic activity from nucleic acid translocation effects. Furthermore, directed evolution strategies (such as the COMPASS system) can lead to enzyme domain truncation due to host expression systems, while bioinformatics predictions (such as AlphaFold2) lack the accuracy to simulate flexible regions and cofactor binding dynamics. These methods are commonly plagued by cross-influencing factors such as metal ion interference, substrate preference, and temperature sensitivity, limiting screening efficiency and industrial application. Therefore, the development of new TdT screening technologies still needs to break through multidisciplinary bottlenecks to promote the application of this enzyme in synthetic biology, gene editing and other fields. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for screening highly active terminal deoxynucleotidyl transferases, and to explore the enzymatic properties of different TdT enzymes in order to discover better starting enzymes for subsequent modification.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: a method for screening highly active terminal deoxynucleotidyl transferase, comprising: (1) Use a known terminal deoxynucleotidyl transferase as a probe to blast the protein database and select homologous enzymes with a sequence identity of 20% or more with the probe or similar protein structure as candidate enzymes; (2) Classify the candidate enzymes according to their species origin, and select several candidate enzymes from each category that have a sequence identity of 20% or more with the probe for enzyme activity analysis; preliminarily screen out the best performing candidate enzymes; (3) The optimal temperature, optimal pH, optimal metal ions, and temperature tolerance of the best performing candidate enzymes screened out are studied to determine the specific conditions that are most conducive to the enzyme's catalytic reaction; and the extension and conversion of the candidate enzyme on different substrates is studied.

[0006] Preferably, in step (2), the species source includes animals, plants or microorganisms.

[0007] Preferably, in step (2), the enzyme activity analysis comprises: heterologously expressing the screened candidate enzyme and purifying the protein; and determining the activity of the screened candidate enzyme in extending dNTP at a reaction temperature of 20-50°C, a reaction time of 10-30 minutes, and in the presence of sufficient substrate.

[0008] Preferably, the activity of the screened candidate enzyme is determined by DNA urea PAGE detection method, fluorescence detection method or by-product pyrophosphate detection method.

[0009] Preferably, in the temperature tolerance study in step (3), the temperature range includes a low temperature of 4°C-20°C and a high temperature of ≥50°C.

[0010] Preferably, in step (3), in the optimal pH study, the pH range is pH 4-10.

[0011] Preferably, in step (3), in the study of the optimal metal ion, the metal ion is selected from Co 2+ Mg 2+ 、Mn 2+ 、Zn 2+ 、Ni + 、Na + , K + 、Li + 、Ag + 、Cd 2+ 、Sr 2 Any one or more of the above; the concentration range of metal ions is 0.1mM-10mM.

[0012] Preferably, in step (3), the substrate includes double-stranded DNA / RNA, single-stranded DNA / RNA, natural dNTP and / or non-natural dNTP.

[0013] The beneficial effects of the present invention are as follows: compared with the prior art, the present invention is simple to operate, does not require expensive detection equipment, and is low in cost; the range of TdT screened is wider, the screened enzyme has better comprehensive performance, and is easier to be transformed into a mutant with stronger performance; and the reaction system obtained in the research can better exert the optimal activity of TdT. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the phylogenetic tree analysis and screening of TdT enzymes in Example 2 of the present invention, including the phylogenetic tree analysis of 226 TdTs. Purple represents Pisces, brown represents Cyclostomata, red represents Reptilia, blue represents Aves, yellow represents Mammalia, and green represents Amphibia. Figure 2 The nine TdTs screened in Example 2 of the present invention with sequence identities between 40.91% and 98.80% with BtTdT and their activity comparisons are shown; wherein, a: sequence identity of the nine TdTs; b: activity comparison of the nine TdTs; As (from Chinese alligator), Bt (from cattle), Ch (from sheep), Cm (from chimaera), Gag (from turtle), Mr (from mouse), Ol (from medaka), Pa (from baboon), Za (from sparrow); Figure 3 This is the purified portion of the TdT enzyme in Example 2 of the present invention; Figure 4 This is the activity detection of some TdT enzymes based on urea DNA-PAGE in Example 2 of the present invention; Figure 5 This is a preliminary analysis of the TdT optimum temperature in Example 2 of the present invention; Figure 6 This is a preliminary analysis of the optimal pH for TdT in Example 2 of the present invention; Figure 7 The optimal reaction conditions of the optimal candidate enzyme BtTdT screened out in Example 2 of the present invention are studied, wherein: a: activity detection of the optimal candidate enzyme by urea DNA-PAGE at different pH values; b: activity detection of the optimal candidate enzyme by urea DNA-PAGE at different temperatures; c: relative activity of the optimal candidate enzyme at different pH values; d: relative activity of the optimal candidate enzyme at different temperatures; e: activity detection of the optimal candidate enzyme by urea DNA-PAGE at different types of metal ions; f: activity detection of the optimal candidate enzyme by urea DNA-PAGE at different concentrations of metal ions; g: relative activity of the optimal candidate enzyme at different types of metal ions; h: optimal temperature; i: optimal Co 2+ Ion concentration. DETAILED DESCRIPTION

[0015] The present invention is described more completely and clearly below with reference to the accompanying drawings and specific embodiments. The embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0016] Example 1 The method for screening highly active terminal deoxynucleotidyl transferase provided in this example has the following specific methods and steps: 1. Phylogenetic tree analysis and screening of TdT enzyme By blasting the UniProt database using mouse-derived MrTdT as a probe, we selected approximately 1,000 different TdT enzymes with sequences identical to MrTdT at 20% or above.

[0017] In one embodiment, the candidate enzyme can also be selected from homologous enzymes that are similar to MrTdT in protein structure, but their sequence identity is not necessarily very high, such as if the sequence identity is less than 30%, that is, it can be other candidate enzymes of the tenth family of DNA polymerases (pol X).

[0018] In addition to animal sources, candidate enzymes can also be selected from plants or microorganisms including bacteria, fungi, viruses, archaea, etc.

[0019] Several TdT enzymes, including the commercial enzyme BtTdT, were screened from different categories according to species origin and sequence identity differences, and were heterologously expressed and purified in Escherichia coli.

[0020] 2. Enzyme Activity Analysis Compare and analyze the activity of these selected TdT enzymes in extending natural or unnatural dNTPs at higher reaction temperatures (e.g., 40°C), longer reaction times (e.g., half an hour), and in the presence of sufficient substrate. Initially screen the best performing TdT enzymes using DNA urea PAGE. The best TdT enzymes exhibit longer or more complete substrate conversion, resulting in brighter and longer bands on PAGE. Alternatively, establish a precise activity assay based on fluorescence detection or byproduct pyrophosphate detection to compare the purified TdT enzyme candidates and select the best performing TdT. For this comparison, maintain the same concentration of each enzyme, and other reaction conditions (e.g., substrate amount, reaction temperature, and reaction time) to more objectively and accurately reflect differences in activity among the candidate enzymes. Bands on the PAGE gel can be quantified using software such as ImageJ, with relative enzyme activity determined based on band grayscale values. Fluorescence signal intensity can be quantified using a fluorescence imaging system or microplate reader to accurately compare and confirm enzyme activity.

[0021] 3. Investigation of the catalytic activity of TdT enzyme The best TdT screened out was studied and analyzed for its optimal temperature, pH, metal ions, temperature tolerance and other reaction conditions to determine the specific conditions that are most conducive to the catalytic reaction of the TdT, so as to support the catalytic activity of TdT to the greatest extent.

[0022] The reaction temperature ranges from low to high temperature, the high temperature can be as high as 50, 60, 70, 80, 90, 100°C or even higher, and the low temperature can be as low as 20, 10, 4°C. The pH range is acidic to alkaline, such as pH4, pH5, pH6, pH7, pH8, pH9, pH10, etc. The metal ions can be common different metal ion compounds, such as Co 2+ / Mg 2+ / Mn 2+ / Zn 2+ / Ni + / Na + / K + / Li + / Ag + Plasma, or less common such as Cd 2+ / Sr 2+ Plasma. The concentration of metal ions can range from as low as 0.1, 0.5 mM to as high as 2, 10 mM. The buffer used in the reaction system can be phosphate buffer, acetate buffer, tris-hydrochloric acid buffer, dimethyl arsenate buffer, etc. Other additives that help improve the performance of enzyme reactions, such as dimethyl sulfoxide (DMSO), sodium chloride, glycerol, and surfactant Tween 20, also need to be optimized and explored in terms of added amount. Explore the optimal TdT extension conversion for different substrates, such as double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), natural dNTPs, and non-natural dNTPs (such as dNTPs with different protecting groups on the 3'-OH).

[0023] Example 2 The method for screening highly active terminal deoxynucleotidyl transferase provided in this example has the following specific methods and steps: 1. Phylogenetic tree analysis and screening of TdT enzyme Using cattle BtTdT as a probe, 226 TdTs were selected from the UniProt database to construct a phylogenetic tree, such as Figure 1 As shown in , the species list: Alligator sinensis (Alligator sinensis), Bos taurus (ox), Capra hircus (sheep), Callorhinchus milii (Chimaera), Gopherus agassizii (turtle), Mus musculus (mouse), Oryzias latipes (Melal fish), Papio Anubis (baboons) and Zonotrichia albicollis (Sparrow). The protein sequences screened showed at least 20% homology to BtTdT. These TdTs came from seven classes within the subphylum Vertebrata, including Cyclostomes, Pisces (Chondrostei and Osteichthyes), Amphibia, Reptiles, Aves, and Mammals.

[0024] From the above classes, 9 TdTs with sequence identity between 40.91% and 98.80% with BtTdT were selected for enzyme activity analysis, such as Figure 2 As shown in a and b. In addition, avian ZaTdT from NCBI was selected as a positive control. Subsequently, its gene was placed into the pETDuet-1 plasmid and transformed into Escherichia coli JM109 (DE3) strain. After heterologous expression and protein purification using Ni-NTA His-Tag purification agarose, TdT with His-Tag was obtained, as shown in Figure 3 shown.

[0025] 2. Enzyme Activity Analysis First, the reaction products were subjected to DNA urea polyacrylamide gel electrophoresis (PAGE) to compare the enzyme activity, and the TdT with the best activity was selected from the nine candidate enzymes for subsequent research and modification. Figure 4 shown.

[0026] The optimal temperature, optimal pH, optimal metal ion and temperature tolerance of the screened TdT with the best activity were analyzed. The results are as follows: Figure 5 、 Figure 6 shown.

[0027] When comparing TdT catalytic activity with natural dNTPs or 3'-modified non-natural dNTPs, the grayscale value of each sample in the PAGE gel can be calculated using ImageJ software, allowing for a more accurate comparative analysis of TdT catalytic activity. The results revealed that BtTdT was the best TdT, exhibiting the highest activity toward natural dNTPs. Furthermore, BtTdT, ChTdT, and ZaTdT exhibited higher conversion rates for 3'-ONH2-dNTPs and 3'-OCN3-dNTPs.

[0028] In summary, BtTdT has better overall performance than other candidate TdTs, making it the optimal TdT selected. Therefore, BtTdT can be used as the starting enzyme for further detailed research, engineering modification, and step-by-step DNA synthesis.

[0029] 3. Exploration of the catalytic activity of BtTdT --The optimal temperature, pH, and metal ions of BtTdT enzyme were preliminarily tested and analyzed using denaturing DNA urea PAGE detection method. ImageJ software was used to quantitatively analyze the gray value of the product (n+m). Figure 7 As can be seen from Figures ad, as the pH value or temperature increases, the activity gradually increases, reaches a peak value, and then gradually decreases. Figure 7 As can be seen in Figure d, at 40℃, the coupling efficiency is increased by about 58% and 22% respectively compared with 20℃ and 30℃ used in other literature. The optimal pH and temperature are pH 7.0 and 40℃ respectively. Figure 7 As shown in c and d. In order to determine the optimal metal ions and corresponding concentrations of the enzyme reaction system, the effects of 11 different positive monovalent or divalent ions on enzyme activity were evaluated. Figure 7 As shown in the example, the results show that Co 2+ / Mg 2+ / Mn 2+ / Zn 2+ The suitable concentration range of the four metal ions was studied to be 0-10 mM with an interval of 2 mM. Figure 7 As shown in f, four metal ions Co 2+ / Mg 2+ / Mn 2+ / Zn 2+ The optimal concentrations of BtTdT were 2 / 4 / 4 / 2 mM. 2+ Concentration. Better Co 2+ As shown, the temperature is about 43 ° C and the concentration is about 1.5 mM. Figure 7 h and i in the middle.

[0030] In summary, the TdT with the best comprehensive activity screened by the method of the present invention is BtTdT from cattle, which has an optimal reaction temperature of about 43°C, an optimal reaction pH of about 7.0, and an optimal ion concentration of 1.5 mM Co 2+ Wild-type BtTdT has high catalytic activity for natural dNTPs and also exhibits high catalytic activity for non-natural fluorescein-labeled or 3'-block dNTPs, such as 3'-ONH2-dNTP, 3'-OCN3-dNTP, 3'-OC2H2CN-dNTP, and heterologous DNA mononucleotides. Therefore, BtTdT can be used as the starting enzyme for the synthetases of these natural or non-natural nucleic acids and further modified to enhance its catalytic performance.

Claims

1. A method for screening highly active terminal deoxynucleotidyl transferase, characterized in that: The following steps are involved: (1) Use a known terminal deoxynucleotidyl transferase as a probe to blast the protein database and select homologous enzymes with a sequence identity of 20% or more with the probe or similar protein structure as candidate enzymes; (2) Classify the candidate enzymes according to their species origin, and select several candidate enzymes from each category that have a sequence identity of 20% or more with the probe for enzyme activity analysis; preliminarily screen out the best performing candidate enzymes; (3) The optimal temperature, optimal pH, optimal metal ions, and temperature tolerance of the best-performing candidate enzymes screened out are studied to determine the specific conditions that are most conducive to the enzyme's catalytic reaction; and the extension and conversion of the candidate enzyme on different substrates is studied.

2. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, wherein: In step (2), the species source includes animals, plants or microorganisms.

3. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, wherein: In step (2), the enzyme activity analysis includes: heterologously expressing the screened candidate enzyme and purifying the protein; and determining the activity of the screened candidate enzyme in extending dNTP at a reaction temperature of 20-50°C, a reaction time of 10-30 minutes, and in the presence of sufficient substrate.

4. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 3, wherein: The activity of the screened candidate enzymes is determined by DNA urea PAGE detection method, fluorescence detection method or by-product pyrophosphate detection method.

5. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, wherein: In step (3), in the optimum temperature study, the temperature range includes low temperature 4°C-20°C and high temperature ≥50°C.

6. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, wherein: In step (3), in the optimal pH study, the pH range is 4-10.

7. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, wherein: In step (3), in the study of the optimal metal ion concentration, the metal ion is selected from Co 2+ Mg 2+ 、Mn 2+ 、Zn 2+ 、Ni + 、Na + , K + 、Li + 、Ag + 、Cd 2+ 、Sr 2 Any one or more of the following; the concentration range of the metal ion is 0.1mM-10mM.

8. The method for screening highly active terminal deoxynucleotidyl transferase according to claim 1, wherein: In step (3), the substrate includes double-stranded DNA / RNA, single-stranded DNA / RNA, natural dNTP and / or unnatural dNTP.

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