Terminal deoxyribonucleotidyl transferase variant, nucleic acid and kit

Abstract

The present invention provides a terminal deoxyribonucleotidyl transferase variant, nucleic acid, and kit, and relates to the field of genetic engineering. The terminal deoxyribonucleotidyl transferase variant comprises a molecular chaperone, a flexible linker, and an amino acid sequence as shown in SEQ ID NO: 2; the molecular chaperone is fused to the N-terminus or C-terminus of the amino acid sequence SEQ ID NO: 2 through the flexible linker. The TDT variant provided by the present disclosure has a high expression yield in Escherichia coli and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a terminal deoxyribonucleotidyl transferase variant, nucleic acid and a kit. Background Art

[0002] Terminal deoxynucleotidyl transferase (TdT) is a special DNA polymerase that can bind deoxynucleotides to the 3'-OH end of DNA molecules independently of the template. It was first purified from mammalian calf thymocyte lysates. With the continuous deepening of research, three subtypes of TdT have been observed in humans and cattle: TdTS, TdTL1 and TdTL2. They all have the activity of adding nucleotides to the available 3'-OH end independently of the template. TdT's unique ability to create genomic materials from scratch makes it one of the most fascinating DNA polymerases in nature. In recent years, research on TdT has made significant progress. As a biological catalyst involved in DNA replication, splicing and repair, it plays an important role in tumorigenesis, developmental processes, and gene expression regulation. In terms of molecular structure and function: through X-ray crystallography and other means, researchers have analyzed the three-dimensional structure of TDT and derived the key binding sites; in the drug development stage: continuously searching for and developing inhibitors of TDT for use in cancer treatment; in terms of function and mechanism of action: continuously exploring the role of TDT in biological processes, DNA damage repair, and cell apoptosis.

[0003] When cells undergo apoptosis, double-strand breaks or single-strand breaks in chromosome DNA produce a large number of sticky 3'-OH ends. Under the action of terminal deoxyribonucleotidyl transferase (TdT), the derivatives formed by deoxyribonucleotides and fluorescein, peroxidase, alkaline phosphatase or biotin can be labeled to the 3'-end of DNA, so that apoptotic cells can be detected. This type of method is called terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). TUNEL is a research method that combines molecular biology and morphology. It can accurately reflect the most typical biochemical and morphological characteristics of apoptosis by in situ staining of intact single apoptotic cell nuclei or apoptotic bodies. It can be used for apoptosis determination in paraffin-embedded tissue sections, frozen tissue sections, cultured cells and cells isolated from tissues, and can detect very small amounts of apoptotic cells. Its sensitivity is much higher than that of general histochemical and biochemical determination methods, so it has been widely used in the study of apoptosis, and TDT is an indispensable component in the study of apoptosis.

[0004] To date, many biological effects of TdT have not been fully revealed. Since the polymerization activity of TdT is independent of the template chain, the molecular details of nucleotide binding and selection remain elusive. The uses of TdT have not yet been fully discovered, but the preparation of TdT has problems such as unstable activity or low yield and cumbersome purification methods, which have become important factors affecting the batch-to-batch differences in subsequent TdT applications and have also created resistance to TdT research.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0006] In view of this, the present invention provides a terminal deoxyribonucleotidyl transferase variant, nucleic acid, and kit, which improve the expression level and enzyme activity of the terminal deoxyribonucleotidyl transferase by performing point mutation on the amino acid sequence of the terminal deoxyribonucleotidyl transferase and modifying the molecular chaperone.

[0007] The technical solution of the present invention is achieved as follows: The present invention provides a terminal deoxyribonucleotidyl transferase variant, which includes a molecular chaperone, a flexible linker and an amino acid sequence as shown in SEQ ID NO: 2; the molecular chaperone is fused to the N-terminus or C-terminus of the amino acid sequence SEQ ID NO: 2 through the flexible linker.

[0008] On the basis of the above technical scheme, preferably, the SEQ ID NO:2 is substituted by glutamic acid at the serine at position 6 corresponding to SEQ ID NO:1, by arginine at the serine at position 25, by alanine at the glycine at position 133, by glutamine at the leucine at position 220, by tryptophan at position 242, by asparagine, and by alanine at the serine at position 265.

[0009] On the basis of the above technical solution, preferably, the amino acid sequence of the molecular chaperone is as shown in SEQ ID NO:11.

[0010] On the basis of the above technical solution, preferably, the amino acid sequence of the molecular chaperone is as shown in SEQ ID NO:13.

[0011] On the basis of the above technical solution, preferably, the flexible linker has a sequence of glycine and serine as repeating modules: (GGGGS)m, wherein m=2 to 5, and m is an integer.

[0012] Based on the above technical solution, preferably, the amino acid sequence of the flexible linker is as shown in SEQ ID NO:9.

[0013] The present invention also provides a nucleic acid encoding the terminal deoxyribonucleotidyl transferase variant.

[0014] The present invention also provides a kit, which comprises the terminal deoxynucleotidyl transferase variant as described above.

[0015] The terminal deoxyribonucleotidyl transferase variant of the present invention has the following beneficial effects compared with the prior art: a mutant terminal deoxyribonucleotidyl transferase is obtained by performing point mutation on the terminal deoxyribonucleotidyl transferase, and then a molecular chaperone is screened to optimize the mutant terminal deoxyribonucleotidyl transferase, thereby obtaining a TDT variant with high expression yield and stable enzyme activity in Escherichia coli, providing stable materials for the application and research of the terminal deoxyribonucleotidyl transferase and increasing the application scenarios of the TDT enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the expression result of bacteria transformed with plasmid A and plasmid B in one embodiment of the present invention.

[0018] Figure 2 This is the expression result of bacteria transformed with plasmid C and plasmid D in one embodiment of the present invention.

[0019] Figure 3 This is the expression result of bacteria transformed with plasmid E and plasmid F in one embodiment of the present invention.

[0020] Figure 4 This is the expression result of bacteria transformed with plasmid G and plasmid H in one embodiment of the present invention.

[0021] Figure 5 This is the TDT test result of the purification process of bacteria transformed with plasmid A in one embodiment of the present invention.

[0022] Figure 6 This is the TDT test result of the purification process of bacteria transformed with plasmid B in one embodiment of the present invention.

[0023] Figure 7This is the TDT test result of the purification process of bacteria transformed with plasmid C in one embodiment of the present invention.

[0024] Figure 8 This is the TDT test result of the purification process of bacteria transformed with plasmid D in one embodiment of the present invention.

[0025] Fig. 9 This is the TDT test result of the purification process of bacteria transformed with plasmid E in one embodiment of the present invention.

[0026] Fig.10 This is a TDT test result of the purification process of bacteria transformed with plasmid F in one embodiment of the present invention.

[0027] Fig.11 This is a schematic diagram comparing the results of flow cytometry quality inspection of the activity of terminal deoxyribonucleotidyl transferase expressed by Escherichia coli corresponding to each plasmid in an embodiment of the present invention.

[0028] Fig.12 This is a schematic diagram of the flow cytometry results of bacteria transformed with plasmid A in one embodiment of the present invention.

[0029] Fig.13 This is a schematic diagram of the flow cytometry results of bacteria transformed with plasmid B in one embodiment of the present invention.

[0030] Fig.14 This is a schematic diagram of the flow cytometry results of bacteria transformed with plasmid C in one embodiment of the present invention.

[0031] Fig.15 This is a schematic diagram of the flow cytometry results of bacteria transformed with plasmid D in one embodiment of the present invention.

[0032] Fig.16 This is a schematic diagram of the flow cytometry results of bacteria transformed with plasmid E in one embodiment of the present invention.

[0033] Fig.17 This is a schematic diagram of the flow cytometry results of bacteria transformed with plasmid F in one embodiment of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] (1) In this example, the amino acid sequence segment Met 127-Ala509 in the terminal deoxyribonucleotidyl transferase was mutated to obtain the TDT-2 amino acid sequence. At the same time, the TDT-2 amino acid sequence was fused with a molecular chaperone to obtain a terminal deoxyribonucleotidyl transferase variant.

[0037] The Met 127-Ala 509 segment in the amino acid sequence of the wild-type terminal deoxyribonucleotidyl transferase was obtained as TDT-1 (SEQ ID NO: 1), and 5 sites in its amino acid sequence were mutated: S (Ser) at position 6 was mutated to E (Glu), S (Ser) at position 25 was mutated to R (Arg), G (Gly) at position 133 was mutated to A (Ala), L (Leu) at position 220 was mutated to Q (Gln), W (Trp) at position 242 was mutated to N (Asn), and S (Ser) at position 265 was mutated to A (Ala) to obtain TDT-2 (mutant) (SEQ ID NO: 2), and the amino acid sequence is shown in the table below (the sites in bold and underlined are the mutated sites).

[0038]

[0039] (2) The molecular chaperones SAPs (short peptide) (SEQ ID NO: 10), IbpA (SEQ ID NO: 11), hspb6 (SEQ ID NO: 12), CRYAB (SEQ ID NO: 13), CIpb (SEQ ID NO: 14), and nusA (SEQ ID NO: 15) were obtained respectively. The specific amino acid sequences are shown in the table below.

[0040]

[0041] (3) TDT-2 was connected to the molecular chaperone using a flexible linker (SEQ ID NO: 9) to obtain a fusion sequence. The fusion sequence includes SAPs-TDT-2 (SEQ ID NO: 3), IbpA-TDT-2 (SEQ ID NO: 4), hspb6-TDT-2 (SEQ ID NO: 5), CRYAB-TDT-2 (SEQ ID NO: 6), CIpb-TDT-2 (SEQ ID NO: 7), and nusA-TDT-2 (SEQ ID NO: 8). The specific amino acid sequences are shown in the table below.

[0042]

[0043]

[0044] Example 2

[0045] In this embodiment, pET-19b is used as a vector, and Nde1 and BamH I are used as restriction sites to synthesize plasmids respectively having synthetic nucleic acids expressing amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 9. Exemplarily, plasmid A has a synthetic nucleic acid expressing an amino acid sequence SEQ ID NO: 1, plasmid B has a synthetic nucleic acid expressing an amino acid sequence SEQ ID NO: 2, plasmid C has a synthetic nucleic acid expressing an amino acid sequence SEQ ID NO: 3, plasmid D has a synthetic nucleic acid expressing an amino acid sequence SEQ ID NO: 4, plasmid E has a synthetic nucleic acid expressing an amino acid sequence SEQ ID NO: 5, plasmid F has a synthetic nucleic acid expressing an amino acid sequence SEQ ID NO: 6, plasmid G has a synthetic nucleic acid expressing an amino acid sequence SEQ ID NO: 7, and plasmid H has a synthetic nucleic acid expressing an amino acid sequence SEQ ID NO: 8.

[0046] Plasmids A, B, C, D, E, F, G and H were transformed into Rosetta DE3 competent cells, inoculated into resistant LB plate medium, and grown overnight. 1-3 single clones on the transformation plate were selected and inoculated into 3mL resistant liquid medium respectively; cultured at 37℃, 220rpm until OD600nm was 0.5-0.6. Take 0.7mL of bacterial solution for glycerol preservation and freezing for later use. Take another bacterial solution and add IPTG with a final concentration of 0.5 mmol / L, induce expression at 28℃ for 4h, centrifuge the bacterial solution after induction of expression to collect the corresponding bacteria of each plasmid transformed bacteria, and ultrasonically disrupt them, and use polyacrylamide gel electrophoresis (SDS-PAGE) to detect the expression of terminal deoxyribonucleotidyl transferase in each bacteria. See the schematic diagram of gel electrophoresis results of plasmids with corresponding amino acid sequences of TDT-1, TDT-2, SAPs-TDT-2, IbpA-TDT-2, hspb6-TDT-2, CRYAB-TDT-2, CIpb-TDT-2, and nusA-TDT-2 for details. Figure 1-Figure 4 , and Table 1.

[0047] like Figure 1 As shown, the expression results of bacteria transformed with plasmid A and plasmid B, wherein electrophoresis lane 1 on the horizontal axis is a protein marker, electrophoresis lanes 2-4 are the terminal deoxyribonucleotidyl transferase expression results of bacteria transformed with plasmid A (three parallel experiments are set up), and electrophoresis lanes 5-7 are the terminal deoxyribonucleotidyl transferase expression results of bacteria transformed with plasmid B (three parallel experiments are set up).

[0048] like Figure 2As shown, the expression results of bacteria transformed with plasmid C and plasmid D, wherein electrophoresis lane 1 of the horizontal axis is a protein marker, electrophoresis lane 2 is the expression result of terminal deoxyribonucleotidyl transferase of bacteria transformed with plasmid C, electrophoresis lane 3 is a blank control, and electrophoresis lane 4 is the expression result of terminal deoxyribonucleotidyl transferase of bacteria transformed with plasmid D.

[0049] like Figure 3 As shown, the expression results of bacteria transformed with plasmid E and plasmid F, wherein electrophoresis lane 1 of the horizontal axis is a protein marker, electrophoresis lanes 2 and 3 are the terminal deoxyribonucleotidyl transferase expression results of bacteria transformed with plasmid E, and electrophoresis lanes 4 and 5 are the terminal deoxyribonucleotidyl transferase expression results of bacteria transformed with plasmid F.

[0050] like Figure 4 As shown, the expression results of bacteria transformed with plasmid G and plasmid H, wherein electrophoresis lanes 1 and 2 on the horizontal axis are the terminal deoxyribonucleotidyl transferase expression results of bacteria transformed with plasmid G, electrophoresis lane 3 is a protein marker, electrophoresis lane 4 is a blank control, and electrophoresis lanes 5 and 6 are the terminal deoxyribonucleotidyl transferase expression results of bacteria transformed with plasmid H.

[0051]

[0052] Combining Table 1 and Figure 1-Figure 4 The protein expression results showed that plasmids A to F all expressed terminal deoxyribonucleotidyl transferase TDT in E. coli, while plasmids G and H did not express terminal deoxyribonucleotidyl transferase TDT in E. coli.

[0053] Deoxyribonucleotidyl transferase TDT.

[0054] Example 3

[0055] According to Example 2, the Escherichia coli expressing TDT can be screened out and cultured amplified, and the bacteria obtained by the amplified culture can be subjected to cell disruption, centrifugation, chromatography, etc., the terminal deoxyribonucleotidyl transferase expressed in the bacterial cells can be extracted from the cells and purified, and then the purified terminal deoxyribonucleotidyl transferase can be activated by enzyme activity assay, electrophoresis, etc.

[0056] (1) Scale-up culture

[0057] Step 1. Take 20 μL of bacteria corresponding to each plasmid transformed by glycerol preservation in Example 2, and inoculate them into 20 mL LB (Amp+) ampicillin-resistant liquid culture medium (wherein the concentration of ampicillin antibiotic solution is 50 μg / mL), shake at 200 rpm at 37°C overnight, and place at room temperature after 15h-17h. Wherein, the liquid LB culture medium is 1% NaCL, 1% tryptone, and 0.5% yeast extract, and is sterilized at 121°C for 30min after preparation.

[0058] Step 2: After standing at room temperature for 7-9 hours, take 1 mL of overnight cultured bacterial solution and transfer it to 500 mL TB (AMP+) ampicillin-resistant liquid medium (wherein the concentration of ampicillin antibiotic solution is 50 μg / mL), add 2.5 mL of 20% glucose sterilized separately to make the final concentration of glucose 0.1%, and then add 520 μL of 1 mol / L MgSO4 (taking the error into account) to make the final concentration of MgSO4 1 mmol / L, and culture overnight at 16°C 200 rpm with shaking. Wherein, the formula of the TB medium includes: 1.2% tryptone, 2.4% yeast extract, 0.9% glycerol, 72 mmol / L K2HPO4, 17 mmol / L KH2PO4, and autoclave at 121°C for 20 minutes.

[0059] Step 3. After culturing for 16 hours, when each group of E. coli reached the logarithmic growth phase (OD600=0.5-0.6), 500 μL of 1 mol / L IPTG (inducer) was added to make the final concentration of 1 mmol / L. After timed induction at 16°C and 200 rpm for 18 hours, the cells were centrifuged at 8000 rpm for 1 min, the bacteria were collected, and stored at -20°C; bacterial sludge expressing TDT-1, TDT-2, SAPs-TDT-2, IbpA-TDT-2, hspb6-TDT-2, and CRYAB-TDT-2 were obtained.

[0060] In step 3, it should be noted that if the E. coli has not reached the logarithmic growth phase after 16 hours of culture, it should be cultured at 37°C with shaking at 200 rpm until OD600nm = 0.5-0.6. If the bacteria cannot be collected in time after IPTG induction, it should be placed in a 16°C shaker.

[0061] (2) Chromatographic separation

[0062] Step 10, crushing and centrifugation: Add 100 mL of resuspension solution to the bacterial sludge expressing TDT-1, TDT-2, SAPs-TDT-2, IbpA-TDT-2, hspb6-TDT-2, and CRYAB-TDT-2, respectively, and fully resuspend all the bacterial sludge to make the bacterial solution uniform. Wherein, the resuspension solution includes 50 mmol / L HEPES, 15 mmol / L KCl, 5 mmol / L MgCl2, 10 mL of glycerol (mass concentration in the resuspension solution is 10%), 10 mmol / L imidazole, 1 mmol / L AEBSF (protease inhibitor), 0.2%-0.5% SKL (sodium lauroyl sarcosine), 1% Triton X-100, and 1 mmol / L EDTA. In this step, in order to prevent protein degradation, the more water-soluble and stable protease inhibitor AEBSF is selected.

[0063] High-pressure homogenizer (Ningbo Xinzhi) 300bar, low-pressure crushing for 3min, then 1000bar, high-pressure crushing for 15min. After low-pressure crushing, high-pressure crushing is performed again to prevent small bacterial particles from clogging the pipeline. After three cycles of high-pressure crushing, when the crushed bacteria are clear and transparent without turbidity, centrifuge at 4°C, 12000rpm, 30min, and collect the supernatant to obtain the corresponding protein supernatant.

[0064] Step 20, balancing the filler: Take out the Ni-NTA filler and wash and balance it with a balancing solution, so that the filler is in the same buffer system as the protein supernatant to be separated, so that the protein can be more fully combined with the filler. The balancing solution includes 20 mmol / L HEPES, pH 7.4, 300 mmol / L NaCl, and 10% glycerol.

[0065] Step 30, centrifugal incubation: Mix the protein supernatant obtained in step 10 with the balanced filler obtained in step 20. For every 100 mL of protein supernatant, add 3 mL of filler beads (Ni-NTA filler, manufacturer Boglon) and 10 mL of glycerol (final mass concentration of 10%), and incubate at 4°C-8°C, shaker at low speed of 20 rpm for 1 hour. In this way, protein aggregation or loss is avoided during the subsequent purification process.

[0066] After incubation, centrifuge at 33 rpm for 5 min, discard the supernatant, and divide the centrifuged filler evenly into four 15 mL gravity columns.

[0067] Step 40, balancing and washing: washing each gravity column with a balancing solution of 5 times the volume of the gravity column, wherein the balancing solution comprises 20 mmol / L HEPES, pH 7.4, 300 mmol / L NaCl, and 10% glycerol.

[0068] The fillers in each gravity column were washed with the first washing solution of 5 to 10 times the volume of the gravity column until the outflowing first washing solution did not turn blue obviously when detected by Coomassie Brilliant Blue G-250.

[0069] Continue to use 5-10 times the volume of the gravity column of the second washing solution to wash the fillers in each gravity column until the outflowing washing solution is not obviously blue when detected by Coomassie Brilliant Blue G-250.

[0070] The first washing solution includes 20 mmol / L HEPES pH 7.4, 300 mmol / L NaCl, 10% glycerol, and 20 mmol / L imidazole. The second washing solution includes 20 mmol / L HEPES pH 7.4, 300 mmol / L NaCl, 10% glycerol, and 50 mmol / L imidazole.

[0071] Step 50, elution purification: add 5-10 mL of the first eluent, allow the eluent to fully combine with the filler at low temperature for 1 hour, then use the first eluent to elute for 2 column volumes, wait until there is no obvious blue color in the G250 detection, collect 3 column volumes of supernatant to obtain the first supernatant. After that, immediately add DTT with a final concentration of 2 mmol / L to the first supernatant and store at low temperature.

[0072] Then add 1 column volume of the second eluent to the filler of each gravity column, incubate at low temperature for 20 minutes, collect the second supernatant, immediately add DTT with a final concentration of 2mmol / L to the second supernatant, and store at low temperature. The first supernatant and the second supernatant need to be mixed to obtain an eluted supernatant. The first eluent includes 20 mmol / L HEPES pH 7.4, 300mmol / L NaCl, 15% glycerol, 500mmol / L imidazole, 0.2%-0.5% (mass fraction) SKL (sodium N-dodecylamine), and 1mmol / L DTT. The second eluent includes 20mmol / L HEPES pH 7.4, 300mmol / L NaCl, 10% glycerol, 500mmol / L imidazole, 2mmol / L DTT, and 0.2%-0.5% SKL.

[0073] A portion of the eluted supernatant was used for SDS-PAGE verification. The verification results are shown in Figure 5-Figure 10 .

[0074] See also Figure 5 and Figure 6, electrophoresis lane 1 on the horizontal axis is the detection result of the protein marker; electrophoresis lane 2 is the detection result of the eluate flowing out of the gravity column; electrophoresis lane 3 is the detection result of the second washing liquid flowing out of the gravity column; electrophoresis lane 4 is the detection result of the first washing liquid flowing out of the gravity column; electrophoresis lane 5 is the detection result of the equilibrium liquid flowing out of the gravity column.

[0075] See also Figure 7 and Figure 8 , electrophoresis lane 1 on the horizontal axis is the detection result of the equilibrium liquid flowing out of the gravity column; electrophoresis lane 2 is the detection result of the first washing liquid flowing out of the gravity column; electrophoresis lane 3 is the detection result of the second washing liquid flowing out of the gravity column; electrophoresis lane 4 is the detection result of the elution liquid flowing out of the gravity column; electrophoresis lane 5 is the protein marker detection result.

[0076] See also Fig. 9 , electrophoresis lane 1 on the horizontal axis is the detection result of the equilibrium liquid flowing out of the gravity column; electrophoresis lane 2 is the detection result of the first washing liquid flowing out of the gravity column; electrophoresis lanes 3 and 4 are the detection results of the second washing liquid flowing out of the gravity column; electrophoresis lane 5 is the detection result of the elution liquid flowing out of the gravity column; electrophoresis lane 6 is the protein marker detection result.

[0077] See also Fig.10 , electrophoresis lane 1 on the horizontal axis is the detection result of the equilibrium solution flowing out of the gravity column; electrophoresis lane 2 is the detection result of the second washing solution flowing out of the gravity column; electrophoresis lane 3 is the detection result of the elution solution flowing out of the gravity column; electrophoresis lane 4 is the protein marker detection result.

[0078] The above steps are protein purification steps. In the above process, HEPES is selected as the buffer system for the purification solution, which has the following effects: 1. Maintaining the stability and activity of the protein to avoid denaturation or inactivation of the TDT enzyme; 2. Providing an ionic environment to regulate the charge properties of the protein, which is helpful for the separation and purification of the protein. 3. Inhibiting protein interactions and improving the efficiency of purification. 4. Regulating protein solubility, which is helpful for the separation and precipitation of proteins. 5. Inhibiting hydrophobic interactions, making it easier to separate proteins. In addition to selecting HEPES, the appropriate pH is also an important condition. The pH of the washing solution and the elution solution were adjusted to 7.4. 5%-10% glycerol was added in each step of the purification process, and 2mmol / L DTT reducing agent was added to the eluted product in real time. Glycerol and DTT can reduce the large-scale aggregation of TDT. After the elution is completed, SDS-PAGE is used to verify the purity, and the subsequent desalting step is immediately performed using a purifier. This measure also reduces the TDT enzyme purification cycle and reduces the inactivation and large-scale aggregation of enzyme proteins under the influence of high concentrations of imidazole. After desalting, the product is collected, the sample concentration and purity are verified, and 50% glycerol and 2mmol / L DTT are added for flow cytometry quality inspection.

[0079] Step 60, desalting and flow cytometry: After the supernatant collected in step 40 is sterilized by filtration through a 0.22 μm filter, the qualified target product is verified and desalted according to the desalting procedure of the purifier. The above desalting is for the corresponding product tubes of each genotype. Among them, the desalting solution / final storage solution: 50mmol / L Tris; 250mmol / L NaCI, pH 7.4; 0.1mmol / L EDTA; 2mmol / L DTT; 0.1% Triton X-100 15mmol / L arginine.

[0080] After desalting, 50% glycerol, 2 mmol / L DTT, and 0.5 mmol / L EDTA were added, and finally SDS-page verification was performed. The yield results are as follows Fig.11 and Table 2. Among them, see Fig.10 , electrophoresis lanes 5 and 6 are the results of SDS-PAGE detection after desalting and concentrating the cells expressing CRYAB-TDT-2. The activity of terminal deoxyribonucleotidyl transferase was then tested by flow cytometry, and the results are shown in Table 3, and Figure 12-Figure 17 .

[0081]

[0082] according to Fig.11 As shown in Table 2, the expression yield of the mutant TDT-2 was significantly increased compared with the expression yield of the wild-type terminal deoxyribonucleotidyl transferase TDT.

[0083] After the terminal deoxyribonucleotidyl transferase TDT of mutant TDT-2 was fused with various molecular chaperones, the expression yield of CRYAB-TDT-2 was greatly increased, reaching up to 60 mg / L.

[0084]

[0085] In Table 3, when TDT enzyme flow cytometry was used to test activity, the positive control selected was the positive control in the Tunel apoptosis detection kit. The positive control concentration was 0.8 mg / mL, and 10 μL was sampled. The sampling of variants was consistent with that of the positive control. Plasmid G and plasmid H were not expressed, so activity was not tested.

[0086] According to Table 3, and Figure 12-Figure 17 The test results show that the flow cytometry activity of CRYAB- TDT-2 is higher than that of the positive control without the need to remove the molecular chaperone. This process can achieve a high yield of 60 mg / L and qualified activity of terminal deoxyribonucleotidyl transferase TDT through one-step affinity purification, which can realize the high yield of terminal deoxyribonucleotidyl transferase TDT in shake flasks and meet the supply and demand of subsequent raw materials.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A terminal deoxyribonucleotidyl transferase variant, characterized in that The variant consists of a molecular chaperone, a flexible linker and an amino acid sequence as shown in SEQ ID NO:2; the molecular chaperone is fused to the C-terminus of the amino acid sequence SEQ ID NO:2 via a flexible linker; the amino acid sequence of the molecular chaperone is shown in SEQ ID NO:11 or SEQ ID NO:

13.

2. The terminal deoxyribonucleotidyl transferase variant according to claim 1, characterized in that The amino acid sequence SEQ ID NO: 2 is obtained by a point mutation of the amino acid sequence SEQ ID NO:

1.

3. The terminal deoxyribonucleotidyl transferase variant according to claim 1, characterized in that The flexible linker has a sequence of glycine and serine as a repeating module: (GGGGS)m, wherein m=2-5, and m is an integer.

4. The terminal deoxyribonucleotidyl transferase variant according to claim 3, characterized in that The amino acid sequence of the flexible linker is shown in SEQ ID NO:

9.

5. A nucleic acid, characterized in that The nucleic acid encodes the terminal deoxyribonucleotidyl transferase variant according to any one of claims 1 to 4.

6. A kit, characterized in that: The kit comprises the terminal deoxynucleotidyl transferase variant according to any one of claims 1 to 4.

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