A method for the prokaryotic expression of recombinant deoxyribonuclease

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a method for preparing recombinant deoxyribonuclease by prokaryotic expression. The method comprises the following steps: S1, connecting a target gene into a multiple cloning site of an expression vector to construct a recombinant expression vector, wherein the target gene comprises an X X A tag coding gene and a deoxyribonuclease coding gene; S2, introducing the recombinant expression vector into E. coli; S3, culturing the recombinant E. coli and inducing expression; and S4, collecting bacterial bodies after centrifugation of the recombinant E. coli culture solution, ultrasonic crushing, centrifugal collection of cell crushing supernatant, and then affinity purification to obtain a deoxyribonuclease fusion protein. The present application fuses the X X A tag with the deoxyribonuclease, adopts a prokaryotic expression system, has high host bacterium activity in the process of inducing expression, realizes high-efficiency expression of DNase, and significantly improves the soluble expression level, and has the advantages of good DNase biological activity and high stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a method for preparing recombinant deoxyribonuclease by prokaryotic expression. BACKGROUND

[0002] Deoxyribonuclease I (DNase I) is a phosphodiesterase that can hydrolyze single-stranded or double-stranded deoxyribonucleic acid (DNA), and it recognizes and cuts phosphodiester bonds to produce single deoxyribonucleotides or single-stranded or double-stranded oligodeoxyribonucleotides with a phosphate group at the 5' end and a hydroxyl group at the 3' end. The activity of DNase I depends on Ca 2+ , and can be activated by divalent metal ions such as Mg 2+ , Mn 2+ , etc. In the presence of Mg 2+ , the enzyme can randomly recognize and cut any site on either strand of double-stranded DNA; and in the presence of Mn 2+ , it can recognize and cut almost the same site on both strands of DNA to produce blunt-end or sticky-end DNA fragments with 1-2 nucleotides protruding. DNase I is a commonly used tool enzyme in molecular biology experiments, and its main use is to remove DNA during in vitro transcription or RNA purification, and it can also be used to remove DNA contamination in proteins, and can be used in pharmaceutical production, and its most important medical application is to reduce the viscosity of pulmonary sticky secretions, i.e. sputum. For example, in the treatment of pneumonia, respiratory obstruction caused by sticky secretions can cause respiratory discomfort, and even cause suffocation and death, and DNase I can help clean the respiratory tract.

[0003] At present, DNase I from bovine, pig, chicken and mouse has been isolated and purified, among which the commercial application of bovine DNase I is the most extensive. Although bovine pancreatic DNase I (Bp-DNase I) can be directly extracted from bovine pancreas, the purification process is complex and limited by raw materials. Therefore, using genetic engineering technology to express Bp-DNase I is the most convenient and economical strategy to efficiently obtain DNase I, but its application also has many limitations. Since DNase has the activity of degrading DNA, the excessive presence of the protein in host cells will have obvious toxicity to the host cells, resulting in greater difficulty in expressing DNase I in recombinant expression, especially in prokaryotic cells. In order to express a specific target protein in prokaryotic cells, especially in Escherichia coli, the general method at present is to insert the nucleotide sequence encoding the target protein into a suitable expression vector, then transform the host bacteria with the recombinant expression vector, obtain the recombinant engineering bacteria, culture the obtained recombinant engineering bacteria under appropriate culture conditions, and separate and purify the expressed target protein. However, for a target protein such as DNase I which is toxic to host cells, using the commonly described recombinant expression method, a small amount of expressed DNase I will kill the host cells, so that the required amount of target product cannot be obtained. Moreover, the expression of DNase I in prokaryotic cells is also prone to form inclusion bodies, resulting in a complex purification process, low yield, and low biological activity of the expressed protein. Therefore, it is of great industrial use and market value to improve the expression amount and expression activity of DNase I in prokaryotic expression system while ensuring the survival of host cells. SUMMARY

[0004] In view of the problems of low expression amount and expression activity and high toxicity to host cells of DNase I expressed by the prokaryotic expression system in the prior art, the present application provides a method for preparing recombinant deoxyribonuclease by prokaryotic expression.

[0005] The present application is specifically implemented by the following technical solutions:

[0006] The first aspect of the present application provides a method for preparing recombinant deoxyribonuclease by prokaryotic expression, comprising the following steps:

[0007] S1, connecting a target gene into a multiple cloning site of an expression vector to construct a recombinant expression vector, wherein the target gene comprises a X X A tag coding gene and a deoxyribonuclease coding gene from upstream to downstream;

[0008] S2, introducing the recombinant expression vector into Escherichia coli to obtain recombinant Escherichia coli;

[0009] S3, culturing the recombinant Escherichia coli and inducing expression to obtain a recombinant Escherichia coli culture solution;

[0010] S4. After centrifuging the culture fluid of the recombinant Escherichia coli, the cells are collected, the cells are disrupted by ultrasonication, the cell supernatant is collected by centrifugation, and then affinity purification is performed to obtain the deoxyribonuclease fusion protein.

[0011] Furthermore, the target gene also includes a purification tag encoding gene, which is located upstream of the XXA tag encoding gene, and the purification tag is selected from at least one of a His tag and a Myc tag.

[0012] Furthermore, the target gene also includes a restriction enzyme cleavage site encoding gene, and the restriction enzyme cleavage site encoding gene is located between the XXA tag encoding gene and the deoxyribonuclease encoding gene.

[0013] Furthermore, the deoxyribonuclease is deoxyribonuclease I, the purification tags are 6×His tags and Myc tags, and the enzyme cleavage site is an enterokinase enzyme cleavage site.

[0014] Furthermore, the nucleotide sequence of the target gene is shown as SEQ ID NO.3.

[0015] Furthermore, in step S1, ligating the target gene into the multiple cloning site of the expression vector includes the following steps: synthesizing the XXA tag encoding gene and the deoxyribonuclease encoding gene, double-digesting the expression vector pET29a with NdeI and EcoRI restriction endonucleases, and then mixing the double-digested expression vector pET29a with the deoxyribonuclease I encoding gene and the XXA tag encoding gene for homologous recombination.

[0016] Furthermore, in step S2, the recombinant expression vector is introduced into Escherichia coli using a heat shock method.

[0017] Furthermore, in step S3, culturing the recombinant E. coli and inducing expression comprises the following steps: inoculating the recombinant E. coli into a liquid LB medium or TB medium supplemented with kanamycin, and culturing until OD 600 When the pH reaches 0.6-0.7, IPTG is added at a final concentration of 0.1-1 mM and the expression is induced at 37° C. for 4-5 hours. Furthermore, the concentration of IPTG is 0.1 mM.

[0018] Furthermore, in step S4, affinity purification to obtain the deoxyribonuclease fusion protein includes the following steps: loading the cell disruption supernatant onto a well-equilibrated nickel column, sequentially washing with a loading buffer and an elution buffer, and collecting the eluate to obtain the deoxyribonuclease fusion protein, wherein the loading buffer formula includes: 20mM Tris-HCl, 0.5M NaCl, 5mM imidazole and 10% glycerol, and the elution buffer formula includes: 20mM Tris-HCl, 0.5M NaCl, 150mM imidazole and 10% glycerol.

[0019] The first aspect of the present invention provides the use of the above-mentioned method for preparing recombinant deoxyribonuclease by prokaryotic expression in preparing deoxyribonuclease.

[0020] The advantages and positive effects of the present invention are:

[0021] The present invention achieves efficient expression of deoxyribonuclease by fusing the XXA tag with deoxyribonuclease using a prokaryotic expression system, and significantly improves the soluble expression level of the deoxyribonuclease. The deoxyribonuclease fusion protein obtained by prokaryotic expression has the advantages of high expression and purification yield, good biological activity and high stability. In addition, during the induced expression process, the activity of the Escherichia coli host bacteria is not affected, and the deoxyribonuclease can be expressed continuously and stably, which is of great significance for the mass production of deoxyribonuclease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. 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 any creative work.

[0023] Figure 1 This is an SDS-PAGE gel electrophoresis diagram of the protein induced to express after E. coli was transformed with plasmid 1 according to an embodiment of the present invention;

[0024] Figure 2 This is an SDS-PAGE gel electrophoresis diagram of the protein induced to express after E. coli was transformed with plasmid 2 according to an embodiment of the present invention;

[0025] Figure 3 This is an SDS-PAGE gel electrophoresis diagram of the protein induced to express after E. coli was transformed with plasmid 3 according to an embodiment of the present invention;

[0026] Figure 4 This is an SDS-PAGE gel electrophoresis diagram of the protein induced to express after E. coli was transformed with plasmid 4 according to an embodiment of the present invention;

[0027] Figure 5 SDS-PAGE gel electrophoresis chart of the protein expressed after inducing the E. coli transformed with the plasmid 5 of the embodiment of the present application;

[0028] Figure 6 SDS-PAGE gel electrophoresis chart of the endonuclease fusion protein purified after inducing the E. coli transformed with the plasmid 1 of the embodiment of the present application;

[0029] Figure 7 Agarose gel electrophoresis chart of the pUC19 plasmid digested by the endonuclease fusion protein purified of the embodiment of the present application;

[0030] Figure 8 Agarose gel electrophoresis chart of the pUC19 plasmid digested by the endonuclease purified of the embodiment of the present application after storing at different temperatures. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in details below with embodiments. The embodiments described herein are only used to explain the present application and not intended to limit the present application.

[0032] According to the information contained in the present application, various changes to the precise description of the present application can be easily made by those skilled in the art without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the present application is not limited to the defined processes, properties or components, as these embodiments and other descriptions are merely illustrative of the specific aspects of the present application. In fact, various changes to the embodiments of the present application that are obvious to those skilled in the art or related fields are encompassed within the scope of the appended claims.

[0033] In order to better understand the present application without limiting the scope of the present application, all numbers expressing quantities, percentages, and other numerical values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the description and claims are approximations that can vary depending upon the desired properties sought to be obtained in the

[0034] In order to make the above-mentioned objectives, features and advantages of the present application more obvious and understandable, the specific embodiments of the present application will be described in details below with reference to the accompanying drawings.

[0035] The present invention provides a method for preparing recombinant deoxyribonuclease by prokaryotic expression, comprising the following steps:

[0036] S1. Constructing a recombinant expression vector: ligate the target gene into the multiple cloning site of the expression vector to construct a recombinant expression vector, wherein the target gene includes, from upstream to downstream, an XXA tag encoding gene and a deoxyribonuclease encoding gene. Thus, the fusion protein expressed by the recombinant expression vector includes, from N-terminus to C-terminus, an XXA tag and a deoxyribonuclease.

[0037] S2. Transformation into Escherichia coli: introducing the recombinant expression vector into Escherichia coli to obtain recombinant Escherichia coli;

[0038] S3, inducing expression: culturing the recombinant E. coli and inducing expression to obtain a recombinant E. coli culture solution;

[0039] S4. Purification of fusion protein: The recombinant E. coli culture solution is centrifuged to collect the cells, which are disrupted by ultrasound. The cell supernatant is collected by centrifugation and then affinity purified to obtain the deoxyribonuclease fusion protein.

[0040] The present invention has been tested in a large number of experiments in the early experimental process and found that the use of signal peptides such as ompA and pelB to guide the expression of deoxyribonuclease (DNase) fusion protein in the cytoplasmic space of Escherichia coli and the use of commonly used soluble tags such as TrxA, GST, and MBP for solubility-aided expression cannot solve the problem of Escherichia coli host bacteria dying and almost no expression of deoxyribonuclease during deoxyribonuclease expression. Even by optimizing the induction expression process and inducer concentration and other detailed factors, the problem of host bacteria dying and low DNase expression cannot be avoided. Only by fusing the XXA tag with the DNase, the efficient expression of the DNase is achieved using a prokaryotic expression system, and the soluble expression amount is significantly improved. The expressed fusion protein has the advantages of high expression amount and purification yield, good biological activity and high stability. In addition, during the induced expression process, the activity of the Escherichia coli host bacteria is not affected, effectively solving the problem of DNase toxicity to the host cell, facilitating the continuous and stable expression of the deoxyribonuclease by the host bacteria, and having important significance for the mass production of deoxyribonuclease.

[0041] Optionally, to facilitate purification, the target gene also includes a purification tag encoding gene located upstream of the XXA tag encoding gene, and the purification tag is selected from at least one of a His tag and a Myc tag. By attaching a His tag to the fusion protein, a highly pure protein can be obtained through a one-step nickel column purification, making purification simple and convenient.

[0042] Optionally, in order to facilitate the subsequent removal of the XXA tag, etc., an enzyme cleavage site is provided between the XXA tag and the deoxyribonuclease, and the enzyme cleavage site is selected from one of an enterokinase cleavage site, a tobacco etch virus protease cleavage site, or a thrombin cleavage site, thereby removing the extra part outside the DNase I by enzymatic excision.

[0043] In a preferred embodiment, the enzyme cleavage site is an enterokinase cleavage site.

[0044] Optionally, the deoxyribonuclease is deoxyribonuclease I (DNase I).

[0045] Optionally, the His tag is a 6×His tag.

[0046] Optionally, the expression vector is pET29a.

[0047] In a preferred embodiment, the nucleotide sequence of the target gene is shown as SEQ ID NO.3.

[0048] Optionally, in step S1, ligating the target gene into the multiple cloning site of the expression vector includes the following steps: synthesizing the XXA tag encoding gene and the deoxyribonuclease encoding gene, double-digesting the expression vector pET29a with NdeI and EcoRI restriction endonucleases, and then mixing the double-digested expression vector pET29a with the deoxyribonuclease I encoding gene and the XXA tag encoding gene for homologous recombination.

[0049] Optionally, in step S2, the method for transforming Escherichia coli adopts a heat shock method.

[0050] Optionally, in step S3, culturing the recombinant E. coli and inducing expression comprises the following steps: inoculating the recombinant E. coli into a liquid LB medium or TB medium supplemented with kanamycin, and culturing until OD 600 When the pH reached 0.6-0.7, IPTG was added at a final concentration of 0.1-1 mM and the expression was induced at 37°C for 4-5 h.

[0051] Optionally, in step S4, affinity purification to obtain the deoxyribonuclease fusion protein includes the following steps: loading the cell disruption supernatant onto a equilibrated nickel column, washing with a loading buffer and an elution buffer in sequence, and collecting the eluate to obtain the deoxyribonuclease fusion protein, wherein the loading buffer formula includes: 20mM Tris-HCl, 0.5M NaCl, 5mM imidazole and 10% glycerol, and the elution buffer formula includes: 20mM Tris-HCl, 0.5M NaCl, 150mM imidazole and 10% glycerol.

[0052] Based on the same inventive concept, another embodiment of the present invention provides the use of the above-mentioned method for preparing recombinant deoxyribonuclease by prokaryotic expression in preparing deoxyribonuclease.

[0053] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0054] 1. Construction of recombinant expression vector and transformation of Escherichia coli

[0055] This example constructs the following recombinant expression vector:

[0056]

[0057]

[0058] Plasmid pET29a was used as a prokaryotic expression vector. The construction process of plasmid 1-2 is as follows:

[0059] 1.1. Synthesis of target gene

[0060] The DNase I encoding gene was obtained by cloning: bovine pancreatic tissue cells were obtained, RNA was extracted using conventional methods, and then reverse transcribed to obtain cDNA, which was amplified using the following primers to obtain the DNase I encoding gene;

[0061] DNase IF: ATGCTGAAAATCGCCGCGT (see SEQ ID NO. 1);

[0062] DNase IR: TTAGGTCAGGGTGACTTCAACC (see SEQ ID NO. 2);

[0063] The XXA tag was synthesized by Universal Biotechnology.

[0064] 1.2. Ligation of target gene with expression vector pET29a

[0065] The expression vector pET29a was double-digested with NdeI and EcoRI restriction endonucleases. The enzyme digestion system included: 3 μg of pET29a, 1.5 μL of NdeI, 1.5 μL of EcoRI, 1 μL of 10× buffer (purchased from Takara Universal Express Enzyme Buffer), and ddH2O to 30 μL. The enzyme digestion was carried out in a 37°C water bath for 2 h. After that, the double-digested expression vector pET29a was mixed with the above-mentioned target gene for homologous recombination to obtain a recombinant product. The homologous recombination enzyme used was the ClonExpress Ultra OneStep Cloning Kit (Cat. No. C115-01). For homologous recombination procedures, please refer to the kit instructions.

[0066] 1.3. Transformation of recombinant plasmid into E. coli

[0067] The above recombinant products were transformed into Escherichia coli top10 competent cells by heat shock method. After that, the strain transformed with plasmid 1 or 2 was spread on solid LB medium plates containing 50 μg / mL kanamycin antibiotics and cultured for a period of time. Single clones were selected and PCR was performed using T7 universal primers to screen for positive clone plaques. Positive clones were inoculated into liquid LB medium and cultured at 37°C and 220 rpm overnight. After that, the plasmids were extracted and sent to the company for sequencing. The correct recombinant plasmid was further transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and positive recombinant E. coli were screened by the same process as above.

[0068] The gene sequence of the recombinant DNase I protein containing Myc and His tags (Myc&His-DNase I) in plasmid 2 is as follows:

[0069]

[0070] Note: The underlined straight line indicates the Myc tag, the underlined wavy line indicates the His tag, the shaded part indicates the enterokinase cleavage site, and the italic part indicates the DNase I gene.

[0071] The gene sequence of the recombinant DNase I protein containing Myc and His tags and XXA tags (Myc&His-XXA-DNaseI) in plasmid 1 is as follows:

[0072]

[0073]

[0074] Note: The underlined straight line indicates the Myc tag, the underlined wavy line indicates the His tag, the underlined dotted line indicates the XXA tag, the shaded part indicates the enterokinase cleavage site, and the italic part indicates the DNase I gene.

[0075] The construction process of plasmids 3-7 is essentially the same as the above steps, except that in plasmid 3-5, the soluble tag XXA in plasmid 2 is replaced with TrxA, MBP, and GST tags, respectively. In plasmid 6-7, the soluble tag XXA in plasmid 2 is replaced with ompA and pelB signal peptide sequences, which are used to direct the DNase I protein to the cytoplasm of E. coli for expression. The TrxA tag gene sequence is shown below:

[0076] atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccg ttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggcc (see SEQ ID NO.5).

[0077] The MBP tag gene sequence is shown below:

[0078]

[0079] GST标签基因序列如下所示:

[0080] atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaa(见SEQ ID NO.7)。

[0081] OmpA信号肽基因序列如下所示:

[0082] atgaaaaagacagctatcgcgattgcagtggcactggcaggtttcgctaccgtcgctcaggct(见SEQID NO.8)

[0083] PelB信号肽基因序列如下所示:

[0084] atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcc (see SEQ ID NO. 9).

[0085] 2. Inducing expression of recombinant E. coli

[0086] The recombinant E. coli was inoculated into a test tube containing 20 mL of liquid LB medium (with 50 μg / mL of kanamycin antibiotic) at 37°C and 220 rpm for 6-8 h until the bacterial liquid concentration reached OD 600 about 0.8-1.0; the culture was transferred into a fresh conical flask containing 200 mL of liquid LB medium or TB medium (with 50 μg / mL of kanamycin antibiotic) at an inoculation amount of 1%, and was fermented at 37°C and 220 rpm until the OD 600 was 0.6-0.7, and then IPTG was added at a final concentration of 0.1 mM to induce expression for 4-5 h.

[0087] After induction, the culture was centrifuged at 4°C and 8000 rpm for 10 min, and the centrifuged bacterial cells were collected, resuspended in 20 mM Tris-HCl (pH 8.0) at a bacterial cell concentration of 0.1 g / mL, and the resuspension was subjected to cell disruption using an ultrasonic cell disruptor, with the ultrasonic disruption conditions being disruption for 3 s, interval of 5 s, total time of 15 min, and power of 400 W, followed by centrifugation at 4°C and 8000 rpm for 10 min, and the cell disruption supernatant and precipitate were collected, respectively, and the cell disruption liquid, cell disruption supernatant and cell disruption precipitate were sampled, respectively, and electrophoresis was performed to detect the induced expression.

[0088] Figures 1-5 The protein induced expression after the plasmids 1-5 were transformed into E. coli is shown, respectively, wherein lane M represents a molecular marker, lane before induction represents the cell disruption liquid before IPTG induction, lane after induction represents the cell disruption liquid after IPTG induction, lane supernatant represents the cell disruption supernatant collected after centrifugation of the cell disruption liquid, and lane precipitate represents the cell disruption precipitate collected after centrifugation of the cell disruption liquid. As can be seen from the figure, the DNase I soluble expression amount cannot be improved without adding or using a conventional soluble tag (TrxA, MBP and GST tag), and the overall expression amount of DNase I is low, and in addition, when the positive E. coli culture and induced expression are performed, it can be observed that a large amount of host bacteria die and a large amount of cell debris is precipitated at the bottom of the culture medium. The DNase I expression guided by the ompA and pelB signal peptides is used, and the plasmids 6-7 have the same situation as the aforementioned plasmids 2-4, and the corresponding results are not repeated here. When only the XXA tag is used to express fusion with DNase I, the DNase I protein band is observed (Figure 1 The soluble protein expression level in the supernatant was significantly improved, and the suitable IPTG concentration for inducing the expression of the fusion protein was about 0.1 mM.

[0089] The DNase I fusion protein expressed by plasmid 1 was purified.

[0090] 3. Purification of the fusion protein

[0091] The positive E. coli was expanded and ultrasonically broken in the manner of step 1.3, and the broken cell supernatant was collected for affinity purification. The purification column and matrix were TA-NiFF (NTA) affinity purification matrix (purchased from Chu Tian Microspheres, item number Y5018). The whole process flow rate was 1.5 mL / min, the linear flow rate was 45 cm / h, and the retention time was 3.3 min. The specific operation was as follows:

[0092] I. Equilibrium: Connect the nickel column to the protein purification instrument, and then flush with purified water for 5 times the column volume, and then flush with binding buffer for 10 times the column volume until the baseline is stable;

[0093] II. Sample loading: Load the collected supernatant into the chromatography column, and start collecting the flow-through when the UV280 starts to rise obviously;

[0094] III. Impurity washing: After sample loading, continue to flush with the sample loading buffer (binding buffer) until the baseline is stable, and then pause the collection;

[0095] VI. Elution: Flush the chromatography column with elution buffer, and start collecting when the UV280 starts to rise, and then stop collecting the elution sample when it drops to stable;

[0096] V. Cleaning: Flush the column with 1M NaCl for at least 3 times the column volume;

[0097] IV. Preservation: Flush with purified water for 5 times the column volume, and then flush with 20% ethanol for 5 times the column volume to preserve the column.

[0098] The binding buffer (pH 7.5) formula includes 20 mM Tris-HCl, 0.5M NaCl, 5 mM imidazole and 10% glycerol; and the elution buffer (pH 7.5) formula includes 20 mM Tris-HCl, 0.5M NaCl, 150 mM imidazole and 10% glycerol.

[0099] The sample supernatant, flow-through and eluate were subjected to electrophoresis, and the results are shown in Figure 6, in which lane M represents a molecular marker, lane S represents a cell breakage supernatant, lane FT represents a flow-through liquid, and lane E represents an eluate (a target protein). As can be seen from the figure, the DNase I fusion protein with high purity is separated from the cell breakage supernatant, the protein concentration is 1.2 mg / mL, and the purity is more than 85%.

[0100] 4. Activity verification of the fusion protein

[0101] DNase I is a deoxyribonuclease that can digest single-stranded or double-stranded DNA. The activity of the protein can be verified by digesting a standard pUC19 plasmid. The definition of enzyme activity unit is that the amount of enzyme required to completely degrade 1 μg of pUC19 plasmid DNA at 37°C for 10 min is defined as 1 unit (U) of activity. According to the DNase I of about 0.1 μg corresponding to 2 U of enzyme activity of Novagen (item number EN401) and Coastal Protein (item number GMP-E127-01A) and other reference materials, the DNase I fusion protein of the present application is diluted to the corresponding concentration (i.e., 2 U, 0.2 U, 0.02 U, 0.002 U and 0.0002 U of enzyme activity) to digest the standard pUC19 plasmid. The specific operation includes: (1) 20 μL of reaction system is added to a 1.5 mL centrifuge tube, respectively, and the reaction system includes: 10x DNase I Reaction Buffer, 1.2 μg of pUC19, 1 μL of DNase I with different dilution multiples, and water is added to make up; (2) 37°C water bath for 10 min; (3) after adding EDTA to a final concentration of 2.5 mM, 65°C reaction for 10 min, and the reaction is terminated. The reaction completed reaction liquid is sampled for electrophoresis, and the results are shown in Figure 7 . In which, lane M represents a molecular marker DL5000 marker, lane pUC19 represents a plasmid without DNase I digestion, and lanes 2 U, 0.2 U, 0.02 U, 0.002 U and 0.0002 U represent the plasmid after digestion corresponding to enzyme activity.

[0102] As can be seen from Figure 7 , the DNase I after purification has strong activity, and after dilution by 100 times, 0.01 U can still normally digest the pUC19 plasmid.

[0103] A certain mass of DNase I is stored at different temperatures for a corresponding time, and then the enzyme activity is verified by the same method as described above, so as to evaluate the stability of the protein. Figure 8The electrophoresis diagram shows the digestion of 1.5 μg of pUC19 plasmid by the DNase I fusion protein of the present invention after storage at different temperatures for a period of time, wherein lane M represents the molecular marker DL5000 marker, lane 1 represents the plasmid without DNase I digestion, lane 2 represents 0.1 μg of DNase I fusion protein stored at -20°C for 3 months, lane 3 represents 0.1 μg of DNase I fusion protein stored at room temperature for 2 months, lane 4 represents 0.05 μg of DNase I fusion protein stored at room temperature for 2 months, lane 5 represents 0.1 μg of DNase I fusion protein stored at -20°C for 1 month, lane 6 represents 0.1 μg of DNase I fusion protein stored at 25°C for 11 days, and lane 7 represents 0.05 μg of DNase I fusion protein stored at 25°C for 11 days. In addition, the DNase I fusion proteins in lanes 2-4 were dissolved in Tris buffer, and the DNase I fusion proteins in lanes 5-7 were dissolved in PB buffer.

[0104] from Figure 8 As can be seen from the figure, the purified DNase I of the present invention has good stability and still has good activity after being placed at room temperature for 1-2 months in different buffers.

[0105] 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 and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a recombinant deoxyribonuclease fusion protein by prokaryotic expression, characterized in that: The following steps are involved: S1. The target gene is ligated into the multiple cloning site of the expression vector to construct a recombinant expression vector. The target gene includes, from upstream to downstream, a purification tag encoding gene, an XXA tag encoding gene, and a deoxyribonuclease encoding gene. The purification tags are a His tag and a Myc tag. The nucleotide sequence of the XXA tag encoding gene is shown in bases 91 to 666 of SEQ ID NO.

3. The nucleotide sequence of the deoxyribonuclease encoding gene is shown in bases 688 to 1467 of SEQ ID NO.

3. The amino acid sequence of the deoxyribonuclease is shown as follows: MLKIAAFNIRTFGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGCSCGNDSFSREPAVVKFSSHSTKVKEFAV ALHSAPSDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADCSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNCTYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLT; S2. introducing the recombinant expression vector into Escherichia coli to obtain recombinant Escherichia coli; S3. Inoculate the recombinant E. coli into liquid LB medium or TB medium supplemented with kanamycin and culture until OD 600 When the pH reached 0.6-0.7, IPTG was added to a final concentration of 0.1 mM and induced for 4-5 h at 37°C to obtain a recombinant E. coli culture solution; S4. After centrifuging the recombinant E. coli culture solution, the cells are collected, the cells are disrupted by ultrasonication, the cell supernatant is collected by centrifugation, and then affinity purification is performed to obtain the recombinant deoxyribonuclease fusion protein.

2. The method for preparing recombinant deoxyribonuclease fusion protein by prokaryotic expression according to claim 1, characterized in that: The target gene further includes an enzyme cleavage site encoding gene, and the enzyme cleavage site encoding gene is located between the XXA tag encoding gene and the deoxyribonuclease encoding gene.

3. The method for preparing recombinant deoxyribonuclease fusion protein by prokaryotic expression according to claim 1, characterized in that: The nucleotide sequence of the target gene is shown in SEQ ID NO.

3.

4. The method for preparing recombinant deoxyribonuclease fusion protein by prokaryotic expression according to claim 1, characterized in that: In step S1, ligating the target gene into the multiple cloning site of the expression vector includes the following steps: The XXA tag encoding gene and the deoxyribonuclease encoding gene were synthesized, and the expression vector pET29a was double-digested with NdeI and EcoRI restriction endonucleases. The double-digested expression vector pET29a was then mixed with the deoxyribonuclease I encoding gene and the XXA tag encoding gene for homologous recombination.

5. The method for preparing recombinant deoxyribonuclease fusion protein by prokaryotic expression according to claim 1, characterized in that: In step S2, the recombinant expression vector is introduced into Escherichia coli using a heat shock method.

6. The method for preparing recombinant deoxyribonuclease fusion protein by prokaryotic expression according to claim 1, characterized in that: In step S4, affinity purification to obtain the recombinant deoxyribonuclease fusion protein comprises the following steps: The cell disruption supernatant is loaded onto a equilibrated nickel column, washed sequentially with a loading buffer and an elution buffer, and the eluate is collected, wherein the loading buffer comprises: 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole and 10% glycerol, and the elution buffer comprises: 20 mM Tris-HCl, 0.5 M NaCl, 150 mM imidazole and 10% glycerol.

7. Use of the method for preparing recombinant deoxyribonuclease fusion protein by prokaryotic expression according to any one of claims 1 to 6 in preparing deoxyribonuclease.

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