Method for expressing and purifying DNase I

Abstract

The invention belongs to the technical field of biology, and relates to a DNase I expression and purification method. The method comprises the following steps: step 1, inoculating a seed solution into a BMGY culture medium according to the inoculum size of 2-5%, and culturing for 14-18 hours; step 2, adding 50% of glycerol into the fermentation liquid to supplement the glycerol, and continuing to ferment for 2-4 hours after the glycerol is supplemented; step 3, methanol feed supplement liquid is added into the fermentation liquid obtained in the step 2 for methanol feed supplement, the methanol feed supplement liquid is a 50% methanol solution containing 10 g / L of yeast extract and 20 g / L of peptone, and the feed supplement time is 96-120 h; and step 4, purifying the fermentation liquor obtained in the step 3. According to the method, a methanol solution containing a yeast extract and peptone is adopted for material supplementing, and the material supplementing speed and mode are adjusted, so that the expression quantity of the target protein DNase I is obviously improved, and the degradation phenomenon of the target protein can be inhibited. The enzyme activity of unit fermentation liquor of the target protein DNase I prepared by adopting the expression and purification method disclosed by the invention can reach about 3 * 10 < 7 > U / L.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for expressing and purifying DNase I. Background Art

[0002] DNase I (deoxyribonuclease I) is a deoxyribonuclease that can digest single-stranded or double-stranded DNA. It recognizes and cleaves phosphodiester bonds to produce monodeoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides 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 treated by divalent metal ions Mg 2+ , Mn 2+ In Mg 2+ In the presence of Mn, the enzyme can randomly recognize and cut any site on either strand of double-stranded DNA; 2+ When present, it recognizes and cuts almost identical sites on both strands of DNA, generating blunt-ended or sticky-ended DNA fragments with 1-2 nucleotide overhangs.

[0003] DNase I is mainly suitable for RNA extraction, in vitro transcription, the removal of DNA in RT-PCR experiments, DNase I imprinting, nick translation, DNA random fragment library construction and other molecular biology experiments. At present, there are two main ways to express DNase I, namely, Escherichia coli expression and yeast expression. Using Escherichia coli as a host bacterium to express DNase I often forms inclusion bodies, which are inactive in themselves, and the correctly folded protein must be obtained after the renaturation process, but the renaturation process of DNase I inclusion bodies is more difficult, and only a very small amount of active DNase I can be obtained, the recovery rate is very low, and the renaturation difficulty is relatively large. Using Pichia pastoris as a host bacterium simplifies the technical route, and the expressed protein can be secreted outside the cell to the culture medium. Compared with the toxic protein expressed intracellularly in Escherichia coli, it is easier to obtain a higher protein expression amount. In the prior art, Pichia pastoris has been successfully used to express DNase I, but the expression of the target protein is basically only carried out at the shake flask level, and there is no clear quantification of the protein expression amount and enzyme activity. At the same time, protein expression is also achieved in a fermenter using Pichia pastoris, but the concentration and purity of the purified protein are not high, and the enzyme activity per unit volume of culture medium is still low.

[0004] Therefore, there is an urgent need for an expression and purification method that can increase the expression level of DNase I and has high activity of the target protease. Summary of the invention

[0005] In order to increase the expression level of DNase I and ensure that the target protein has high enzyme activity, the present application provides a method for expressing and purifying DNase I.

[0006] The DNase I expression and purification method of the present application adopts the following technical scheme: In one aspect, the present application provides a method for expressing and purifying DNase I, the method comprising the following steps: Step 1, inoculating the seed solution containing the DNase I expression strain into BMGY medium at an inoculation rate of 2%-5%, and culturing for 14-18h; Step 2, adding 50% glycerol to the fermentation broth for glycerol feeding, and continuing the fermentation for 2-4 hours after the feeding is completed; Step 3, adding methanol feeding solution to the fermentation broth obtained in step 2 for methanol feeding, wherein the methanol feeding solution is a 50% methanol solution containing 8-12 g / L yeast extract and 20-25 g / L peptone, and the feeding time is 96-120 h; Step 4: purifying the fermentation broth obtained in step 3.

[0007] In some embodiments, the yeast extract content in the methanol feed solution is 9-12 g / L, 10-12 g / L, preferably, 9 g / L, 10 g / L, 11 g / L, 12 g / L, and more preferably 10 g / L.

[0008] In some embodiments, the content of peptone in the methanol feed solution is 16-24 g / L, 18-22 g / L, 20-21 g / L, preferably, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, more preferably 20 g / L.

[0009] By adopting the above technical scheme, by adding 50% methanol solution containing 8-12g / L yeast extract and 15-25g / L peptone, sufficient nutritional conditions can be provided for the strain during the fermentation process, and the degradation of the target protein is significantly inhibited. Compared with the prior art, the expression amount of the target protein DNase I obtained by fermentation is significantly improved; at the same time, the enzyme activity of the target protein DNase I unit fermentation liquid is also significantly improved, reaching 3×10 7 U / L.

[0010] Furthermore, the seed solution is prepared by inoculating the constructed DNase I expression strain into BMGY culture medium, and culturing at 28-30° C. and 160-220 rpm for 20-26 hours.

[0011] Furthermore, the strain is Pichia pastoris or Escherichia coli, preferably Pichia pastoris.

[0012] By adopting the above technical scheme, a higher specific enzyme activity and protein expression amount can be obtained by using Pichia pastoris to secrete and express DNase I enzyme compared with using Escherichia coli to express DNase I enzyme.

[0013] Furthermore, the fermentation temperature is 28-30°C, pH is 5-6, the ventilation volume is 0.5-2VVM, and the rotation speed is 200-1000rpm.

[0014] Furthermore, the feeding in step 2 is specifically feeding at a rate of 5-10 ml / (L·h) for 6-8 hours.

[0015] Furthermore, in step 3, the feeding is specifically carried out at a rate of 2-4 ml / (L·h) for 12-16 h, and then the rate is increased to 4-8 ml / (L·h) and maintained until the feeding is completed.

[0016] By adopting the above technical scheme, during the expression of the target protein, Pichia pastoris first grows using the nutrients in the culture medium, and after the glycerol in the culture medium is consumed, the dissolved oxygen value in the culture medium rises rapidly, and glycerol is fed. After the glycerol feeding is stopped, the dissolved oxygen value of the culture medium further rises, and Pichia pastoris is in a hungry state. At this time, a methanol feeding solution containing yeast extract and peptone is added for feeding. The inventors found that a too fast feeding speed will reduce the specific activity of DNAse I, and a too slow feeding speed will affect the protein expression amount. After optimization, the feeding speed is set to 2-4ml / (L·h), maintained for 12-16h, and then the feeding speed is increased to 4-8ml / (L·h), maintained until the end of fermentation, and a high expression amount and enzyme specific activity can be obtained.

[0017] Furthermore, a pretreatment step is included before step 4, and the pretreatment step is specifically as follows: adding 1M calcium chloride solution to the fermentation broth obtained in step 3, stirring while adding, and adjusting the pH to 7-8, standing at room temperature for 25-35 minutes, and then centrifuging to obtain the supernatant.

[0018] Furthermore, the volume ratio of the calcium chloride solution to the fermentation broth is (0.5-1.5):10.

[0019] By adopting the above technical solution, adding calcium chloride to the fermentation broth can significantly improve the clarity of the fermentation broth after centrifugation, while not affecting the subsequent enrichment process of column chromatography.

[0020] Furthermore, the purification step is to centrifuge the fermentation broth to remove the supernatant, resuspend the precipitate using 50% AMS, centrifuge again to remove the supernatant, resuspend the precipitate using Ni balance buffer, perform column chromatography, and collect the eluted sample.

[0021] On the other hand, the present application also provides a target protein DNase I expressed and purified by the above method, wherein the enzyme specific activity of the target protein DNase I is 3×10 7 U / L.

[0022] In summary, the present application has the following technical effects compared with the prior art: 1) The present application provides a method for the expression and purification of DNase I. The method uses a methanol solution containing yeast extract and peptone for feeding, and adjusts the feeding speed and method, which can significantly increase the expression amount of the target protein DNase I and also significantly inhibit the degradation of the target protein. The enzyme activity of the target protein DNase I per unit fermentation broth obtained by the expression and purification method of the present application is about 3×10 7 U / L.

[0023] 2) The method provided in the present application can express the target protein DNase I enzyme in high amounts in a fermenter, has high production efficiency, is simple and convenient to operate, and can well meet market demand. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings.

[0025] Figure 1 Schematic diagram of the pPic9K vector used in Example 1.

[0026] Figure 2 The following is the SDS-PAGE electrophoresis result of the shake flask supernatant protein in Example 2.

[0027] Figure 3 The above is the result of Ni column purification of the fermentation broth supernatant in Example 3.

[0028] Figure 4 The concentration of the purified sample in Example 3 is measured.

[0029] Figure 5 This is the purity electrophoresis diagram of the purified sample in Example 3.

[0030] Figure 6 The results of DNAseI enzyme activity assay.

[0031] Figure 7 This is the Ni column purification result after optimization of purification conditions.

[0032] Figure 8 The target protein content in the supernatant of the fermentation tank with different culture media, where the loading volume of lanes 1, 4, and 7 is 5 μL, the loading volume of lanes 2, 5, and 8 is 10 μL, and the loading volume of lanes 3, 6, and 9 is 20 μL.

[0033] Fig. 9 These are the purification results of the target protein obtained by fermentation in fermenters with different culture media. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals or conditions recommended by manufacturers.

[0035] The composition of the culture medium used in the experiment is as follows: YPD medium formula: 1% Yeast Extract, 2% Peptone, 2% Dextrose (glucose); BMGY medium formula: 10g / L yeast extract, 20g / L peptone, 10g / L glycerol, 3g / L K2HPO4, 11.8g / L KH2PO4, 13.4g / L YNB, 0.4mg / L biotin; BSM inorganic salt medium formula: 85% H3PO4 26.7ml / L, CaSO4·2H2O 0.93g / L, K2SO418.2g / L, MgSO4·2H2O 14.9g / L, KOH 4.13g / L, glycerol 40g / L, PMT1 4.0ml / L, 100% ammonia water to adjust pH 5.0 (1 bottle of 50ml plus 800ul ammonia water) or 10g / L ammonium sulfate to adjust pH 5.0; PMT1 (1L) formula: CuSO4·5H2O 6.0g / L, KI 0.088g / L, MnSO4·H2O 3.0g / L, Na2MoO4·2H2O0.2g / L, H3BO3 0.02g / L, CoCl2·6H2O 0.5g / L, ZnCl2 20.0g / L, FeSO4·7H2O 65.0g / L, Biotin0.2g / L, concentrated H2SO4 5.0M.

[0036] The purification buffer formula used in the chromatography column during the experiment is as follows: Ni regeneration: 20 mM Tris-HCl, 300 mM NaCl, 1.5 M imidazole, pH 7.5 ± 0.1; Ni balance: 20 mM Tris-HCl, 100 mM NaCl, 20 mM imidazole, 0.2 mM DTT (added as needed), 10% glycerol, pH 7.5 ± 0.1; Ni high salt: 20 mM Tris-HCl, 1.5 M NaCl, 0.2 mM DTT (added immediately after use), 20 mM imidazole, 10% glycerol, pH 7.5 ± 0.1; Ni elution: 20 mM Tris-HCl, 100 mM NaCl, 500 mM imidazole, 10% glycerol, 0.2 mM DTT (added immediately before use), pH 7.5±0.1.

[0037] Example 1 Construction of DNase I expression strain The nucleotide sequence of the DNase I encoding gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2, which was synthesized by Anhui General Biotechnology Co., Ltd. The expression vector used was the pPic9K vector. Figure 1 As shown. The DNase I fragment was cloned into the pPic9K vector (the vector can be found in Figure 1 ), linearized the plasmid, electroporated into Pichia pastoris competent GS115, coated on SCD plates, and screened for histidine-deficient strains. Several single clones were selected, diluted and spotted on 2 g / L G418 plates, and strains with excellent growth were selected for subsequent DNase I protein fermentation.

[0038] Example 2 Shake flask expression of DNase I 1. The DNase I expression strain selected in Example 1 was inoculated into BMGY medium and cultured at 30° C. and 200 rpm for 24 h.

[0039] 2. Add 2% methanol as inducer, add 2% methanol every 24 hours, and repeat 4 times.

[0040] 3. Take the fermentation liquid of induced expression, centrifuge at 12000rpm for 10min, take the supernatant for SDS-PAGE electrophoresis, the concentration of concentrated gel is 4%, the concentration of separation gel is 12.5%, the sample and loading buffer are mixed in a ratio of 1:1, react in boiling water bath for 5min for loading electrophoresis. Set the initial voltage of the electrophoresis instrument to 120V, increase the voltage to 230V when the sample moves to the separation gel, and end the electrophoresis when the sample moves to the bottom of the electrophoresis tank.

[0041] The SDS-PAGE electrophoresis results of the supernatant were as follows: Figure 2 As shown, the supernatants at 0d, 1d, 2d, 3d, and 4d were taken for sample preparation. The molecular weight of the target protein DNase I was 32kDa. Since the molecular weight of the glycosylated product of the target protein in yeast expression would be larger, there were obvious bands at 37kDa in each lane of the electrophoresis diagram, and they gradually accumulated over time. The results showed that the target protein DNaseI was successfully secreted and expressed.

[0042] Example 3 Purification of DNase I from shake flask fermentation supernatant 1. Take the fermentation liquid in Example 2 and centrifuge it at 12000rpm for 10min, remove the supernatant, add 50% AMS, mix thoroughly until a white precipitate appears in the supernatant, centrifuge it again at 12000rpm for 10min, discard the supernatant, and resuspend the precipitate with Ni balance buffer; connect the resuspended liquid to the chromatography column, and select Ni NTABeads (Tian Di Ren He) as the chromatography filler. The specific steps of chromatography are as follows: 1) Take a Ni-NTA pre-packed column of corresponding specifications according to the required loading capacity, regenerate the pre-packed column using (Ni regeneration) for 1CV, and balance the column using (Ni balance) for 2CV; 2) Load the resuspended sample, control the flow rate, keep the sample retention time on the column greater than 3 minutes, collect the flow-through into a centrifuge tube, and use (Ni balance) to balance the chromatography column after loading for 3CV; 3) Rinse the column with (Ni high salt) for 2CV and collect the flow-through into a centrifuge tube; 4) Equilibrate the column using (Ni balance) until the absorbance of the flow-through at 280 nm is close to the baseline; 5) Use (Ni elution) to elute and collect the elution sample until the absorbance of the flow-through at 280 nm is close to the baseline; 6) Column material post-treatment: Use (Ni regeneration) to regenerate the pre-packed column for 2CV, and finally wash the column material with 5 volumes of ddH2O, add 20% ethanol protective solution, and store at 2-8°C.

[0043] Collect the eluted samples as Figure 3 As shown, sam, ft and p represent the injected sample, the flow-through sample and the eluted sample respectively, and the results show that the sample is completely eluted.

[0044] 2. Use the BCA protein concentration determination kit (C503021) to determine the concentration of the eluted sample. The specific steps are as follows: Use 2mg / ml BSA as the standard to establish a standard curve, with a detection range of 125-2000μg / ml. Perform gradient dilutions on the samples to be tested to ensure that the detection value is within the detection range. Prepare an ELISA plate, add 25μL / well of the diluted standard and sample, and 200μL / well of the BCA working solution. Make two parallels for each sample; shake for 30s to fully mix, seal the ELISA plate, and incubate at 37℃ for 30min. After the ELISA plate is cooled to room temperature, use an ELISA reader to read the absorbance value at 562nm.

[0045] Subtract the absorbance of the blank standard from the absorbance of each standard and the sample to be tested, and calculate the average value; draw a standard curve of the standard, such as Figure 4 The protein concentration of the samples to be tested was calculated using the standard curve.

[0046] The results of the test are as follows Figure 4 The test results are shown in Table 1.

[0047] Table 1 Sample concentration test values Sample name OD value Detection value (mg / ml) DNAseI (diluted 2 times) 0.566 0.72 DNAseI (diluted 4 times) 0.326 0.34 3. Purity determination SDS-PAGE method: After induction, samples were taken every 24 hours, and the supernatant was subjected to SDS-PAGE electrophoresis. A 12% PAGE gel was prepared by self-made method, and the preparation method was referred to the "General Technical Requirements of Part IV of the 2020 Edition of the Chinese Pharmacopoeia: 0541 Electrophoresis Method". The sample to be tested was diluted to 1 mg / ml, 20 μL of the sample was taken and then added to an equal volume of 2x loading buffer. A sample with qualified purity was taken as a control, diluted to 1 mg / ml, 20 μL of the sample was taken and then added to an equal volume of 2x loading buffer, and heated at 100 ° C for 10 min; electrophoresis was performed, 5 μL Maker was loaded on the first lane, followed by the control and the sample to be tested. The control and the sample to be tested were loaded with 1 μg, 2 μg, 5 μg, and 10 μg, respectively. After the loading was completed, electrophoresis was performed at 120V for 50 min. After the electrophoresis was completed, photos were taken for result analysis.

[0048] The results are as follows Figure 5 As shown, the DNase I band of the purified target protein is clear, and there is almost no degradation of the target protein.

[0049] Example 4 Determination of DNase I specific activity Definition of enzyme specific activity: One unit refers to the amount of enzyme required to digest 1 μg of pUC19 fragment in 20 μl of total reaction system at 37°C within 10 minutes.

[0050] Prepare the reaction system: 2 μl 0.5 μg / μl pUC19, 2 μl 10×rCutSmart TM Buffer, add different dilution gradients of DNase I enzyme, and make up to 20 μl with purified water; react in a metal bath at 37°C for 10 min and then detect.

[0051] The nucleic acid gel electrophoresis was used to detect the reaction system samples. The sample volume was 10 μl and the voltage was 120 V. The electrophoresis was terminated when the sample moved to the bottom of the electrophoresis tank.

[0052] The enzyme activity of the shake flask purified sample in Example 3 was tested. The enzyme amounts added were 10ng, 5ng, 2ng, 1ng, 0.1ng, and 0.01ng, respectively. The results showed that Figure 6 As shown, when at least 1ng DNase I is added, 1μg pUC19 can be completely decomposed, and the specific activity of DNase I of the target protein is calculated to be 1000U / ug.

[0053] Example 5-9 DNase I fermenter high density fermentation 1. The DNase I Pichia pastoris expression strain selected in Example 1 was inoculated into 200 ml YPD medium and cultured at 30° C. and 1200 rpm for 26 h.

[0054] 2. Inoculate the seed liquid into a fermenter containing 3L BMGY medium at an inoculum volume of 3%, with a fermentation temperature of 30°C, pH 5.5, aeration volume of 0.5-2VVM, and a rotation speed of 600rpm for 16h.

[0055] 3. Add 50% glycerol to the fermenter at a feed rate of 8 ml / (L·h) for 7 h, and then continue fermentation for 3 h.

[0056] 4. Add 100% methanol solution containing M1 yeast extract and M2 peptone to the fermenter at a feeding rate of V1 for feeding T1, then increase the feeding rate to V2 and maintain until the end of fermentation. The methanol induction time lasts for 100 hours. The two feeding rates are expressed as V1 ml / (L·h) and V2 ml / (L·h), the feeding time is expressed as T1 h, and the concentration of yeast extract and the concentration of peptone are expressed as M1 g / L and M2 g / L, respectively. The condition parameters of different embodiments are shown in Table 2 below.

[0057] Table 2 Indexes of Step 4 in Examples 5-9 <![CDATA[M1]]> <![CDATA[M2]]> <![CDATA[V1]]> <![CDATA[T1]]> <![CDATA[V2]]> Example 5 10 20 3 15 6 Example 6 8 15 2 12 4 Example 7 12 25 4 16 8 Example 8 10 20 3 12 4 Example 9 10 20 4 16 8 5. The fermentation broth was purified according to the purification method in Example 3.

[0058] Example 10 The difference between Example 10 and Example 5 is that, before step 5, a pretreatment step of the fermentation broth is further included: 1M calcium chloride solution is added to the fermentation broth at a volume ratio of 10%, stirring while adding, and the pH is adjusted to 7.5. After standing at room temperature for 30 minutes, the supernatant is obtained by centrifugation.

[0059] Comparative Example 1: Inorganic salt The difference between Comparative Example 1 and Example 5 is that the fermentation medium in step 2 is BSM inorganic salt medium, the methanol feed solution in step 3 is 1000 ml methanol + 12 ml PMT1, and the other steps are the same as Example 5.

[0060] Comparative Example 2: BMGY The difference between Comparative Example 2 and Example 5 is that the methanol feed solution in step 3 is 100% methanol without any components, and the other steps are the same as Example 5.

[0061] Comparative Example 3: No optimization of feeding method and speed The difference between Comparative Example 3 and Example 5 is that the feeding method in step 4 is to continue feeding at a feeding rate of 5 ml / (L·h) until the fermentation is completed, and the other steps are the same as Example 5.

[0062] Target protein DNase I expression and enzyme activity determination The expression level and enzyme specific activity of the protein DNase I obtained by fermentation in Examples 5-10 and Comparative Examples 1-3 were measured using the methods of Examples 3 and 4. The measurement results are shown in Table 3.

[0063] Table 3 Protein expression performance test results of Examples 5-10 and Comparative Examples 1-3 Comparing the test results of Example 5 and Example 10, by optimizing the pretreatment method, adding calcium chloride to the fermentation broth in the fermenter can significantly improve the clarity of the fermentation broth after centrifugation, simplifying the pretreatment process, and at the same time not affecting the subsequent enrichment process of column chromatography, and can be directly enriched by nickel column, which greatly reduces the cost. The purity of the target protein DNase I after purification is as follows: Figure 7 As shown, lane 1 is the purification result of Example 5, and lane 2 is the purification result of Example 10. It can be clearly seen that the purity of the target protein DNase I after pre-treatment is higher.

[0064] Comparison of the fermentation tank supernatant protein content of Example 5 and Comparative Examples 1-3 shows that, as shown in Table 3 and Figure 8As shown, in Comparative Example 1, inorganic salts were used as fermentation medium, and the expression level of the target protein DNase I obtained was 10 mg / L; although Comparative Example 2 used BMGY as fermentation medium, its feed solution was a methanol solution without any components, and the expression level of the target protein DNase I obtained by fermentation was 15 mg / L; Comparative Example 3 used both BMGY as fermentation medium and methanol fermentation broth containing yeast extract and peptone as feed solution, but the feeding method was not optimized; although the expression level of the target protein DNase I in Comparative Example 3 was improved compared with Comparative Examples 1 and 2, it was still significantly lower than the expression level of the target protein DNase I of Example 5, which was 30 mg / L. Therefore, the present application uses BMGY as a fermentation medium to effectively increase the expression level of the target protein DNase I, and uses a methanol fermentation broth containing yeast extract and peptone as a feeding liquid to provide sufficient nutrition for the fermentation strain, thereby significantly increasing the expression level of the target protein DNase I, and further improving the enzyme specific activity. The special feeding method of regulating the feeding speed in stages can also significantly increase the expression level of the target protein DNase I and effectively ensure a high enzyme specific activity.

[0065] Comparing the results of DNase I purification of the target protein in the fermentation tank of Example 5 and Comparative Examples 1-3, it can be seen that Fig. 9 As shown, no matter whether inorganic salt or BMGY is used as the fermentation medium, the target protein DNase I is severely degraded. Therefore, the fermentation method of the present application can also significantly inhibit the degradation of the target protein DNase I.

[0066] Comparison of the test results of Examples 5-10 and Comparative Examples 1-3 shows that the expression level of the target protein DNase I obtained by fermentation in Examples 5-10 is at least 24 mg / L, and the enzyme specific activity can reach about 1×10 6 U / mg, and the degradation of the target protein DNase I was significantly inhibited, and there was almost no degradation of the target protein; while the expression level of the target protein DNase I obtained by fermentation in Comparative Examples 1-3 was low, the enzyme specific activity was not high, and the target protein DNase I also showed serious degradation. Therefore, it is shown that the present application uses BMGY as the fermentation medium, and uses methanol containing yeast extract and peptone as the feed liquid, and regulates the feed rate in stages, so as to obtain DNase I with high expression level and high enzyme specific activity.

[0067] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for expressing and purifying DNase I, characterized in that: The method comprises the following steps, Step 1, inoculate the seed solution containing the DNase I expression strain into BMGY medium at an inoculation rate of 2%-5%, and culture for 14-18 hours; Step 2, adding 50% glycerol to the fermentation broth for glycerol feeding, and continuing the fermentation for 2-4 hours after the feeding is completed; Step 3, adding methanol feeding solution to the fermentation broth obtained in step 2 for methanol feeding, wherein the methanol feeding solution is a 50% methanol solution containing 8-12 g / L yeast extract and 15-25 g / L peptone, and the feeding time is 96-120 h; Step 4: purifying the fermentation broth obtained in step 3.

2. The method according to claim 1, characterized in that The method for preparing the seed solution is to inoculate the constructed DNase I expression strain into BMGY culture medium, and culture at 28-30° C. and 160-220 rpm for 20-26 hours.

3. The method according to claim 2, characterized in that The strain is Pichia pastoris.

4. The method according to claim 1, characterized in that: Fermentation temperature is 28-30℃, pH is 5-6, ventilation volume is 0.5-2VVM, and rotation speed is 200-1000rpm.

5. The method according to claim 1, characterized in that The feeding in step 2 is specifically carried out at a rate of 5-10 ml / (L·h) for 6-8 hours.

6. The method according to claim 1, characterized in that The feeding in step 3 is specifically carried out at a rate of 2-4 ml / (L·h) for 12-16 hours, and then the rate is increased to 4-8 ml / (L·h) and maintained until the feeding is completed.

7. The method according to claim 1, characterized in that Before step 4, a pretreatment step is also included. Specifically, the pretreatment step is to add 1M calcium chloride solution to the fermentation liquid obtained in step 3, stir while adding, adjust the pH to 7-8, stand at room temperature for 25-35 minutes, and then centrifuge to obtain the supernatant.

8. The method according to claim 7, characterized in that The volume ratio of the calcium chloride solution to the fermentation broth is (0.5-1.5):

10.

9. The method according to claim 1, characterized in that: The purification step is to centrifuge the fermentation broth to remove the supernatant, resuspend the precipitate with 50% AMS, centrifuge again to remove the supernatant, resuspend the precipitate with Ni balance buffer, perform column chromatography, and collect the eluted sample.

10. The target protein DNase I expressed and purified by the method described in any one of claims 1 to 9 is characterized in that: The target protein DNase unit fermentation broth enzyme activity is about 3×10 7 U / L.

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