Method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction

Abstract

The invention discloses a method for producing human acetaldehyde dehydrogenase 2. The method comprises the following steps: firstly, respectively inserting a human acetaldehyde dehydrogenase 2 gene and a fusion expression tag NusA gene into a plasmid pET30a (+) to construct a recombinant expression vector pET30a (+)-NusA-ALDH2; and carrying out heat shock conversion to escherichia coli for culture, collecting thalli, extracting plasmids, sequencing, and obtaining the recombinant engineering bacteria if the recombinant engineering bacteria are correct. And finally, carrying out inoculated culture on the recombinant engineering bacteria, adding 0.5-1.5 mM of lactose, carrying out induced expression at 15-25 DEG C and 150 r / min for 16-20 hours, centrifugally collecting thalli, suspending a buffer solution, crushing cells, centrifugally taking the crushed supernatant to obtain NusA-ALDH2 crude enzyme, and purifying by using a nickel column affinity chromatography. The method can be used for mass production of soluble humanized acetaldehyde dehydrogenase 2 from an escherichia coli system.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and particularly relates to a method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression with lactose induction. Background Art

[0002] Human liver mitochondrial aldehyde dehydrogenase 2 (ALDH2) participates in the degradation of ethanol in the human body. After ethanol enters the human body, it first generates acetaldehyde under the catalysis of alcohol dehydrogenase (ADH), and then, with the help of cofactor NAD + With the participation of, acetaldehyde dehydrogenase 2 can effectively hydrolyze acetaldehyde into acetic acid, reducing the harm of acetaldehyde to the human body.

[0003]

[0004] There is a mutant ALDH2*2 of acetaldehyde dehydrogenase 2, in which the 487th glutamic acid mutates to lysine, and its activity is basically lost. 8% of the world's population carries the ALDH2*2 gene, and the number of people carrying the ALDH2*2 gene in East Asia is as high as 560 million. Due to the lack of active acetaldehyde dehydrogenase 2 in these people, acetaldehyde cannot be completely metabolized in the human body. Acetaldehyde stimulates the capillaries on the face to dilate, causing blushing; acetaldehyde may also affect the normal function of the nervous system and cause headache symptoms; at the same time, acetaldehyde may irritate the gastrointestinal tract and cause vomiting. In addition, the lack of acetaldehyde dehydrogenase 2 activity is also related to alcoholic liver disease, cancer, cardiovascular disease, etc.

[0005] In order to deeply explore the role and mechanism of acetaldehyde dehydrogenase 2 in aldehyde metabolism, we need to establish a complete system to produce soluble acetaldehyde dehydrogenase 2. The Escherichia coli expression system is widely used due to its advantages such as fast growth, low cost and high expression level. However, in the process of expressing acetaldehyde dehydrogenase 2 in Escherichia coli, relevant researchers found that acetaldehyde dehydrogenase 2 mostly formed inclusion bodies. The formation of this inclusion body makes it difficult for us to directly obtain active acetaldehyde dehydrogenase 2, which to some extent hinders the study of acetaldehyde dehydrogenase 2. Therefore, it is of great theoretical and practical significance to study the mass production of soluble human acetaldehyde dehydrogenase 2 in the Escherichia coli system. Summary of the invention

[0006] The present invention aims to provide a method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression with lactose induction, so as to realize large-scale production of soluble human acetaldehyde dehydrogenase 2 from an Escherichia coli system.

[0007] A method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression with lactose induction in this scheme comprises the following steps:

[0008] Step 1, construction of recombinant expression vector: insert the human acetaldehyde dehydrogenase 2 gene into the plasmid pET30a(+) through the BamH I and Xho I restriction enzyme sites, and insert the fusion expression tag NusA gene into the plasmid pET30a(+) through the Nde I and Kpn I restriction enzyme sites. The resulting recombinant expression vector is named pET30a(+)-NusA-ALDH2;

[0009] Step 2, culturing recombinant engineered bacteria: transforming the recombinant expression vector into Escherichia coli by heat shock method for culturing, collecting the cultured bacteria and extracting the plasmid pET30a(+)-NusA-ALDH2 and sequencing, and the bacteria with correct sequencing are recombinant engineered bacteria;

[0010] Step 3, purification of NusA-ALDH2: inoculate and culture the recombinant engineered bacteria, then add lactose with a final concentration of 0.5-1.5 mM, and induce expression at 15-25°C and 150 r / min for 16-20 hours; after the induction, collect the bacteria by centrifugation, suspend the bacteria with buffer, and then crush the cells with a cell crusher, collect the crushed supernatant by centrifugation to obtain the NusA-ALDH2 crude enzyme solution; purify NusA-ALDH2 by nickel column affinity chromatography, NusA-ALDH2 is the human acetaldehyde dehydrogenase 2.

[0011] Furthermore, the Escherichia coli is selected from Escherichia coli Rosetta (DE3).

[0012] Furthermore, when the recombinant expression vector is transformed into Escherichia coli by the heat shock method and cultured, an LB solid plate containing 45-55 μg / mL kanapencillin is used for screening; a single clone is picked from the screening plate and inoculated into LB liquid culture medium, kanapencillin with a final concentration of 45-55 μg / mL is added, and cultured at 35-40°C and 180-220 r / min for 10-15 hours.

[0013] Furthermore, when the recombinant engineered bacteria are inoculated and cultured, the recombinant engineered bacteria are inoculated into LB liquid culture medium, kanapencillin with a final concentration of 45 to 55 μg / mL is added, and cultured at 35 to 40° C., 180 to 220 r / min for 10 to 15 hours, and then transferred to fresh LB liquid culture medium, kanapencillin with a final concentration of 45 to 55 μg / mL is added, and cultured at 35 to 40° C., 180 to 220 r / min for 0.8 to 1.5 hours.

[0014] Furthermore, the buffer is a Lysis buffer.

[0015] Furthermore, when the recombinant engineered bacteria are transferred to fresh LB liquid culture medium, lactose is added for induction after culturing for 1 hour.

[0016] Furthermore, the final concentration of lactose is 1 mM.

[0017] Furthermore, the lactose induction temperature was 20°C.

[0018] Furthermore, the lactose induction time was 20 h.

[0019] Further, the specific steps of purifying NusA-ALDH2 using nickel column affinity chromatography are as follows:

[0020] (1) Nickel column equilibration: Add 5 column volumes of Lysis buffer to the nickel column and allow gravity to flow out to equilibrate the column;

[0021] (2) Loading: Add the crude NusA-ALDH2 enzyme solution to the equilibrated nickel column and let it flow out by gravity;

[0022] (3) Washing: Add 10 column volumes of washing buffer (50 mM Tris, 300 mM NaCl, 30 mM imidazole) to the nickel column and allow gravity flow to wash out the impurities bound to the nickel column;

[0023] (4) Elution: Add 2 column volumes of Elution buffer (50 mM Tris, 300 mM NaCl, 300 mM imidazole) to the nickel column and allow gravity to flow out. At this time, the target protein is displaced and eluted, and this portion of the eluate is collected;

[0024] (5) Dialysis: The eluate was transferred to a 14 kD dialysis bag, which was placed in a 20 mM Tris-HCl buffer and dialyzed overnight to reduce the imidazole concentration in the enzyme solution.

[0025] Beneficial effects:

[0026] 1. This patent obtains soluble acetaldehyde dehydrogenase 2 by means of fusion expression, eliminating the tedious and complicated renaturation work and simplifying the purification of acetaldehyde dehydrogenase 2.

[0027] 2. This patent uses cheap lactose as an inducer to induce the expression of the acetaldehyde dehydrogenase 2 gene, which is not only inexpensive but also non-toxic to cells.

[0028] 3. Using lactose as an inducer allows induction immediately after the transformation is completed, without the need to culture the cells to the logarithmic growth phase. This feature simplifies the operating process while effectively saving valuable time costs, providing technical support for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram for constructing a recombinant expression vector;

[0030] Figure 2 This is the electrophoresis diagram of the fusion expression of human acetaldehyde dehydrogenase 2 and NusA;

[0031] Figure 3 This is the electrophoresis diagram of human acetaldehyde dehydrogenase 2 fused with NusA after purification by nickel column;

[0032] Figure 4 This is the curve of acetaldehyde oxidation catalyzed by NusA-ALDH2;

[0033] Figure 5 This is the electrophoresis diagram of the effect of induction temperature on the fusion expression of human acetaldehyde dehydrogenase 2 and NusA;

[0034] Figure 6 The electrophoresis diagram shows the effect of the concentration of inducer lactose on the fusion expression of human acetaldehyde dehydrogenase 2 and NusA;

[0035] Figure 7 The electrophoresis diagram shows the effect of lactose induction time on the fusion expression of human acetaldehyde dehydrogenase 2 and NusA;

[0036] Figure 8 This is the electrophoresis diagram of the effect of lactose induction time on the fusion expression of human acetaldehyde dehydrogenase 2 and NusA;

[0037] Fig. 9 This is the electrophoresis diagram showing the effect of disruption time on the target protein content in the supernatant. DETAILED DESCRIPTION

[0038] The following is further described in detail through specific implementation methods:

[0039] Example 1: A method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction

[0040] The following steps are involved:

[0041] Step 1: Construction of recombinant expression vector

[0042] 1. Sequence analysis of human aldehyde dehydrogenase 2

[0043] The sequences of the human acetaldehyde dehydrogenase 2 encoding gene SEQ ID NO1 and the fusion expression tag NusA encoding gene SEQ ID NO3 in the present invention are as follows:

[0044] The sequence of SEQ ID NO1 is as follows:

[0045] TCAGCCGCCGCCACCCAGGCCGTGCCTGCCCCCAACCAGCAGCCCGAGGTCTTCTGCAACCAGATTTTCATAAAC

[0046] AATGAATGGCACGATGCCGTCAGCAGGAAAACATTCCCCACCGTCAATCCGTCCACTGGAGAGGTCATCTGTCAGGTAG

[0047] CTGAAGGGGACAAGGAAGATGTGGACAAGGCAGTGAAGGCCGCCCGGGCCGCCTTCCAGCTGGGCTCACCTTGGCGCCG

[0048] CATGGACGCATCACACAGGGGCCGGCTGCTGAACCGCCTGGCCGATCTGATCGAGCGGGACCGGACCTACCTGGCGGCC

[0049] TTGGAGACCCTGGACAATGGCAAGCCCTATGTCATCTCCTACCTGGTGGATTTGGACATGGTCCTCAAATGTCTCCGGT

[0050] ATTATGCCGGCTGGGCTGATAAGTACCACGGGAAAACCATCCCCATTGACGGAGACTTCTTCAGCTACACACGCCATGA

[0051] ACCTGTGGGGGTGTGCGGGCAGATCATTCCGTGGAATTTCCCGCTCCTGATGCAAGCATGGAAGCTGGGCCCAGCCTTG

[0052] GCAACTGGAAACGTGGTTGTGATGAAGGTAGCTGAGCAGACACCCCTCACCGCCCTCTATGTGGCCAACCTGATCAAGG

[0053] AGGCTGGCTTTCCCCCTGGTGTGGTCAACATTGTGCCTGGATTTGGCCCCACGGCTGGGGCCGCCATTGCCTCCCATGA

[0054] GGATGTGGACAAAGTGGCATTCACAGGCTCCACTGAGATTGGCCGCGTAATCCAGGTTGCTGCTGGGAGCAGCAACCTC

[0055] AAGAGAGTGACCTTGGAGCTGGGGGGGAAGAGCCCCAACATCATCATGTCAGATGCCGATATGGATTGGGCCGTGGAAC

[0056] AGGCCCACTTCGCCCTGTTCTTCAACCAGGGCCAGTGCTGCTGTGCCGGCTCCCGGACCTTCGTGCAGGAGGACATCTA

[0057] TGATGAGTTTGTGGAGCGGAGCGTTGCCCGGGCCAAGTCTCGGGTGGTCGGGAACCCCTTTGATAGCAAGACCGAGCAG

[0058] GGGCCGCAGGTGGATGAAACTCAGTTTAAGAAGATCCTCGGCTACATCAACACGGGGAAGCAAGAGGGGGCGAAGCTGC

[0059] TGTGTGGTGGGGGCATTGCTGCTGACCGTGGTTACTTCATCCAGCCCACTGTGTTTGGAGATGTGCAGGATGGCATGAC

[0060] CATCGCCAAGGAGGAGATCTTCGGGCCAGTGATGCAGATCCTGAAGTTCAAGACCATAGAGGAGGTTGTTGGGAGAGCC

[0061] AACAATTCCACGTACGGGCTGGCCGCAGCTGTCTTCACAAAGGATTTGGACAAGGCCAATTACCTGTCCCAGGCCCTCC

[0062] AGGCGGGCACTGTGTGGGTCAACTGCTATGATGTGTTTGGAGCCCAGTCACCCTTTGGTGGCTACAAGATGTCGGGGAG

[0063] TGGCCGGGAGTTGGGCGAGTACGGGCTGCAGGCATACACTGAAGTGAAAACTGTCACAGTCAAAGTGCCTCAGAAGAAC

[0064] TCA

[0065] The sequence of SEQ ID NO3 is as follows:

[0066]

[0067] The amino acid sequence of human acetaldehyde dehydrogenase 2 SEQ ID NO2 and the amino acid sequence of fusion expression tag NusA SEQ ID NO4 of the present invention are as follows:

[0068] SEQ ID NO2 sequence is as follows:

[0069] SAAATQAVPAPNQQPEVFCNQIFINNEWHDAVSRKTFPTVNPSTGEVICQVAEGDKEDVDKAVKAARAAFQLGSP

[0070] WRRMDASHRGRLLNRLADLIERDRTYLAALETLDNGKPYVISYLVDLDMVLKCLRYYAGWADKYHGKTIPIDGDFFSYT

[0071] RHEPVGVCGQIIPWNFPLLMQAWKLGPALATGNVVVMKVAEQTPLTALYVANLIKEAGFPPGVVNIVPGFGPTAGAAIA

[0072] SHEDVDKVAFTGSTEIGRVIQVAAGSSNLKRVTLELGGKSPNIIMSDADMDWAVEQAHFALFFNQGQCCCAGSRTFVQE

[0073] DIYDEFVERSVARAKSRVVGNPFDSKTEQGPQVDETQFKKILGYINTGKQEGAKLLCGGGIAADRGYFIQPTVFGDVQD

[0074] GMTIAKEEIFGPVMQILKFKTIEEVVGRANNSTYGLAAAVFTKDLDKANYLSQALQAGTVWVNCYDVFGAQSPFGGYKM

[0075] SGSGRELGEYGLQAYTEVKTVTVKVPQKNS

[0076] The sequence of SEQ ID NO4 is as follows:

[0077] MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVRVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRITTQTAKQVIVQKVREAER AMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILREDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEIKAAARDPGSRAKIAVKTNDKRIDP VGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTVDDLQAKHQAEAHAAIDTFTKYLDIDEDFAT VLVEEGFSTLEELAYVPMKELLEIEGLDEPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDLAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA

[0078] 2. Construction of recombinant expression vector

[0079] According to the sequences of human acetaldehyde dehydrogenase 2 encoding gene SEQ ID NO1 and fusion expression tag NusA encoding gene SEQ ID NO3 (commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis), human acetaldehyde dehydrogenase 2 encoding gene SEQ ID NO1 and fusion expression tag NusA encoding gene were connected to plasmid pET30a(+), specifically: human acetaldehyde dehydrogenase 2 gene was inserted through Bam HI and Xho I restriction enzyme sites, and fusion expression tag NusA gene was inserted through Nde I and Kpn I restriction enzyme sites, and the obtained recombinant expression vector was named pET30a(+)-NusA-ALDH2 (see Appendix Figure 1 ).

[0080] Step 2: Cultivation of recombinant engineered bacteria

[0081] The above recombinant expression vector was transformed into Escherichia coli Rosetta (DE3) by heat shock method, and screened using LB solid plates containing 50 μg / mL kanapencillin. Pick a single clone on the screening plate and inoculate it into LB liquid culture medium, add kanapencillin with a final concentration of 50 μg / mL, and culture it at 37°C, 200r / min for 12h. After the culture, collect the bacteria, use a plasmid extraction kit to extract the plasmid pET30a(+)-NusA-ALDH2, and send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Select the bacteria with correct sequencing (recombinant engineered bacteria) and store them in glycerol tubes. The recombinant engineered bacteria are named Rosetta-pET30a(+)-NusA-ALDH2.

[0082] Step 3: Purification of NusA-ALDH2

[0083] The recombinant engineered bacteria Rosetta-pET30a(+)-NusA-ALDH2 stored in the glycerol tube was inoculated into LB liquid medium, and kanamycin with a final concentration of 50 μg / mL was added. The culture was continued at 37°C 200r / min for 12h, and then transferred to fresh LB liquid medium, kanamycin with a final concentration of 50 μg / mL was added, and the culture was continued at 37°C 200r / min for 1h. The inducer lactose with a final concentration of 1mM was added, and the expression was induced at 20°C 150r / min for 20h. After the induction, the cells were collected by centrifugation, and the cells were suspended in Lysis buffer (50mM Tris, 300mM NaCl, 10mM imidazole), and then the cells were broken by a cell disruptor. The cell disruptor conditions were: break for 2s, stop for 5s, and the total time was 15min. The supernatant was collected by centrifugation at 12000r / min to obtain the NusA-ALDH2 crude enzyme solution.

[0084] NusA-ALDH2 was purified by nickel column affinity chromatography. The specific steps are as follows:

[0085] (1) Nickel column equilibration: Add 5 column volumes of Lysis buffer to the nickel column and allow gravity to flow out to equilibrate the column;

[0086] (2) Loading: Add the above-mentioned NusA-ALDH2 crude enzyme solution to the above-mentioned balanced nickel column and let it flow out by gravity. At this time, NusA-ALDH2 contains a histidine tag and binds with nickel ions with affinity and is adsorbed to the nickel column;

[0087] (3) Washing: Add 10 column volumes of washing buffer (50 mM Tris, 300 mM NaCl, 30 mM imidazole) to the nickel column, let gravity flow out to wash out the impurities bound to the nickel column, and use Coomassie Brilliant Blue to detect the washing effect;

[0088] (4) Elution: Add 2 column volumes of Elution buffer (50 mM Tris, 300 mM NaCl, 300 mM imidazole) to the nickel column and allow gravity to flow out. At this time, the target protein is displaced and eluted, and this portion of the eluate is collected;

[0089] (5) Dialysis: The eluate was transferred to a 14 kD dialysis bag, which was placed in a 20 mM Tris-HCl buffer and dialyzed overnight to reduce the imidazole concentration in the enzyme solution, thereby obtaining a purified NusA-ALDH2 enzyme solution, i.e., human acetaldehyde dehydrogenase 2.

[0090] Expression was detected by SDS-PAGE

[0091] The results of fusion expression of acetaldehyde dehydrogenase and NusA are shown in Figure 2 Lane 1 is the whole cell before induction, lane 2 is the whole cell after induction, M is the protein marker, lane 3 is the supernatant of cell disruption after induction, and lane 4 is the precipitate of cell disruption after induction. Figure 2 It can be seen that the protein NusA-ALDH2 expressed by the fusion of human acetaldehyde dehydrogenase 2 and NusA is between 100-135kD, and the theoretical value of the relative molecular weight of NusA-ALDH2 is 112kD, which is consistent with expectations. Further observation shows that NusA-ALDH2 is mainly concentrated in the supernatant of the crushing, and the content in the crushing precipitate is relatively small, indicating that the NusA tag can successfully promote the soluble expression of human acetaldehyde dehydrogenase 2 in Escherichia coli.

[0092] The protein purification was checked by SDS-PAGE.

[0093] The results of NusA-ALDH2 purification are shown in Figure 3 , M is a protein marker, and lane 1 is the purified NusA-ALDH2. The electrophoresis diagram shows that after purification, NusA-ALDH2 presents a relatively single band, and the band size is between 100-135 kD, which is consistent with the expected size.

[0094] NusA-ALDH2 catalyzes acetaldehyde oxidation

[0095] The following ingredients were mixed: 0.3 mL of 1 mol / L Tris-HCl (pH 8.5), 0.05 mL of 20 mmol / L β-NAD, 0.05 mL of 100 mmol / L acetaldehyde, 0.1 mL of 3 mol / L KCl, 0.03 mL of 1 mol / L β-mercaptoethanol, and 2.37 mL of H2O, and added to a cuvette. Finally, 0.10 mL of NusA-ALDH2 was added, and the absorbance at 340 nm was measured at regular intervals. NAD +It is a coenzyme in the catalytic reaction of ALDH2. In the catalytic reaction, NAD + It accepts hydrogen and becomes NADH. NADH has a maximum absorption value at 340nm. Therefore, the amount of NADH generated can be calculated by calculating the change in absorbance at 340nm within a specific time.

[0096] Depend on Figure 4 It can be seen that as the reaction time increases, OD 340 nm It shows an increasing trend, indicating that as time goes by, the NADH in the reaction system increases, that is, the fusion protein NusA-ALDH2 has the activity of human acetaldehyde dehydrogenase 2 and can catalyze the conversion of acetaldehyde into acetic acid.

[0097] The purification parameters of NusA-ALDH2 were determined as follows:

[0098] 1. Induction temperature

[0099] The glycerol-preserved recombinant engineered bacteria Rosetta-pET30a(+)-NusA-ALDH2 was inoculated into LB liquid medium, and kana penicillin with a final concentration of 50 μg / mL was added. The cells were cultured at 37°C 200 r / min for about 12 hours, and then transferred to fresh LB liquid medium according to 1% of the inoculation volume, and kana penicillin with a final concentration of 50 μg / mL was added. The cells were cultured at 37°C 200 r / min for about 2.5 hours, and the inducer IPTG with a final concentration of 1 mM was added. The expression was induced at 16°C, 20°C, 30°C, and 37°C for 6 hours. After the induction, the cells were collected by centrifugation, suspended in 1×PBS buffer, and then the cells were broken by a cell disruptor. The cell disruptor conditions were: power 60%, break 2s, stop 5s, and total time 15min. After the crushing is completed, the supernatant and precipitate are separated by centrifugation, and the expression is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The induction expression temperature for subsequent experiments is determined based on the content of NusA-ALDH2 in the supernatant;

[0100] Figure 5This is an electrophoresis diagram of the effect of induction temperature on the expression of human acetaldehyde dehydrogenase 2 and NusA fusion. Lane 1 is the whole cell before induction, lanes 2-5 are the whole cells after induction at 16℃, 20℃, 30℃, and 37℃, respectively, M is a protein marker, lanes 6-9 are the supernatants of cell fragmentation after induction at 16℃, 20℃, 30℃, and 37℃, respectively, and lanes 10-13 are the precipitates of cell fragmentation after induction at 16℃, 20℃, 30℃, and 37℃, respectively. It can be seen from the electrophoresis diagram that with the increase of the induction temperature, the content of NusA-ALDH2 in the supernatant of fragmentation shows a trend of first increasing and then decreasing. The main reason for this phenomenon is that within a certain temperature range, with the increase of temperature, the expression rate of the gene is correspondingly accelerated, but when the temperature is high, the rate of protein synthesis is too fast, resulting in the newly synthesized protein not having time to fold into the correct spatial structure and easily forming inclusion bodies. It can be seen from the electrophoresis diagram that under the induction condition of 20°C, the content of NusA-ALDH2 in the supernatant was the highest, so the subsequent induction temperature was determined to be 20°C.

[0101] 2. Concentration of inducer lactose

[0102] According to the above-mentioned recombinant engineering bacteria induction expression steps, the induction was carried out at a final concentration of 1mM IPTG and a final concentration of 0.25mM, 0.5mM, 1mM, 2mM, and 4mM lactose, respectively, and the induction temperature was 20°C. The type and concentration of the inducer for subsequent experiments were determined according to the content of NusA-ALDH2 in the supernatant;

[0103] Figure 6 This is an electrophoretic diagram of the effect of the concentration of inducer lactose on the fusion expression of human acetaldehyde dehydrogenase 2 and NusA. Lane 1 is the whole cell before induction, lane 2 is the whole cell after induction with a final concentration of 1mM IPTG, lanes 3-7 are the whole cells after induction with final concentrations of 0.25mM, 0.5mM, 1mM, 2mM, and 4mM lactose, M is a protein marker, lane 8 is the supernatant of cell disruption after induction with a final concentration of 1mM IPTG, and lanes 9-13 are the supernatants of cell disruption after induction with final concentrations of 0.5mM, 1mM, 2mM, and 4mM lactose. It can be seen from the electrophoretic diagram that with the increase of lactose concentration, the content of NusA-ALDH2 in the whole cell shows a trend of first increasing and then decreasing. Among them, the content of NusA-ALDH2 in the whole cell is the highest when the lactose concentration is 1mM, and the content of NusA-ALDH2 in the supernatant of cell disruption at this concentration is basically the same as that when IPTG is induced. In view of the advantages of lactose being low in price and non-toxic to cells compared with IPTG, lactose was determined to be the inducer with a final concentration of 1 mM.

[0104] 3. Lactose induction timing

[0105] According to the above-mentioned recombinant engineering bacteria induction expression steps, the glycerol bacteria were cultured in LB medium at 37°C and 200 r / min overnight, and then transferred to fresh LB liquid medium. After culturing at 37°C and 200 r / min for 0 h, 1 h, 2.5 h, and 4.0 h, lactose with a final concentration of 1 mM was added and induced at 20°C. The induction timing of subsequent experiments was determined according to the content of NusA-ALDH2 in the supernatant.

[0106] Figure 7 The electrophoresis diagram shows the effect of lactose induction time on the expression of human acetaldehyde dehydrogenase 2 and NusA fusion; lane 1 is the whole cell before induction, lanes 2-5 are the whole cells after induction at 0h, 1h, 2.5h, and 4.0h, respectively, M is the protein marker, lanes 6-9 are the supernatants of cell fragmentation after induction at 0h, 1h, 2.5h, and 4.0h, respectively, and lanes 10-13 are the precipitates of cell fragmentation after induction at 0h, 1h, 2.5h, and 4.0h. It can be seen from the electrophoresis diagram that even if lactose is added immediately after the transfer of bacteria for induction, NusA-ALDH2 can still be expressed in large quantities in a soluble form. Compared with the induction with lactose added immediately after the transfer, the content of NusA-ALDH2 in the supernatant was relatively higher when lactose was added 1h, 2.5h, and 4.0h after the transfer. However, the content of NusA-ALDH2 in the supernatant was basically the same among the three groups with the induction time of 1h, 2.5h, and 4.0h. Considering the time cost, the induction time was selected as 1h after the transfer.

[0107] 4. Induction duration

[0108] According to the above-mentioned recombinant engineering bacteria induction expression steps, 1 hour after transfer, lactose with a final concentration of 1mM was added, and the induction was carried out at 20°C. The induction time was 5h, 10h, 20h, and 25h respectively. The induction time of subsequent experiments was determined according to the content of NusA-ALDH2 in the supernatant;

[0109] Figure 8 This is an electrophoresis diagram of the effect of lactose induction time on the fusion expression of human acetaldehyde dehydrogenase 2 and NusA. Lane 1 is the whole cell before induction, lanes 2-5 are the whole cells after induction for 5h, 10h, 20h, and 25h, respectively. M is a protein marker, lanes 6-9 are the supernatants of cell fragmentation after induction for 5h, 10h, 20h, and 25h, respectively, and lanes 10-13 are the precipitates of cell fragmentation after induction for 5h, 10h, 20h, and 25h. It can be seen from the electrophoresis diagram that due to the hysteresis of lactose induction, when the induction time is 5h and 10h, the content of NusA-ALDH2 in the whole cell and the supernatant is very small. After 20h and 25h of induction, the content of NusA-ALDH2 in the whole cell and the supernatant is equivalent. Based on time cost considerations, the induction time is selected to be 20h.

[0110] 5. Broken Time

[0111] According to the above-mentioned steps of inducing expression of recombinant engineered bacteria, 1L of bacterial solution was prepared. One hour after transfer, lactose with a final concentration of 1mM was added, and the induction was carried out at 20°C for 20 hours to obtain the induced recombinant engineered bacteria. The induced recombinant engineered bacteria were broken for 5min, 10min, 15min, 20min, 25min, and 30min respectively. The breaking time for subsequent experiments was determined according to the content of NusA-ALDH2 in the supernatant.

[0112] Fig. 9 The electrophoresis diagram shows the effect of the fragmentation time on the amount of supernatant enzyme; M is a protein marker, lanes 1-6 are the fragmentation supernatants with fragmentation time of 5min, 10min, 15min, 20min, 25min, and 30min, respectively, and lanes 7-12 are the fragmentation precipitates with fragmentation time of 5min, 10min, 15min, 20min, 25min, and 30min, respectively. The electrophoresis diagram shows that when the fragmentation time is 15min, the content of NusA-ALDH2 in the fragmentation supernatant is already high, and the content of NusA-ALDH2 in the fragmentation precipitate is reduced. Considering that the heat generated during fragmentation may affect the enzyme activity, the fragmentation time is selected to be 15-20min.

[0113] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression with lactose induction, characterized in that: The following steps are involved: Step 1, construction of recombinant expression vector: insert the human acetaldehyde dehydrogenase 2 gene into the plasmid pET30a(+) through the Bam HI and Xho I restriction enzyme sites, and insert the fusion expression tag NusA gene into the plasmid pET30a(+) through the Nde I and Kpn I restriction enzyme sites. The resulting recombinant expression vector is named pET30a(+)-NusA-ALDH2; Step 2, culturing recombinant engineered bacteria: transforming the recombinant expression vector into Escherichia coli by heat shock method for culturing, collecting the cultured bacteria and extracting the plasmid pET30a(+)-NusA-ALDH2 and sequencing, and the bacteria with correct sequencing are recombinant engineered bacteria; Step 3, purification of NusA-ALDH2: inoculate and culture the recombinant engineered bacteria, then add lactose with a final concentration of 0.5-1.5 mM, and induce expression at 15-25°C and 150 r / min for 16-20 hours; after the induction, collect the bacteria by centrifugation, suspend the bacteria with buffer, and then crush the cells with a cell crusher, collect the crushed supernatant by centrifugation to obtain the NusA-ALDH2 crude enzyme solution; purify NusA-ALDH2 by nickel column affinity chromatography, NusA-ALDH2 is the human acetaldehyde dehydrogenase 2.

2. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 1, characterized in that: The Escherichia coli is selected from Escherichia coli Rosetta (DE3).

3. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 2, characterized in that: When the recombinant expression vector is transformed into Escherichia coli by the heat shock method for culture, LB solid plates containing 45-55 μg / mL kanapencillin are used for screening; a single clone is picked from the screening plate and inoculated into LB liquid culture medium, kanapencillin with a final concentration of 45-55 μg / mL is added, and cultured at 35-40° C. and 180-220 r / min for 10-15 hours.

4. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 3, characterized in that: When the recombinant engineered bacteria are inoculated and cultured, the recombinant engineered bacteria are inoculated into LB liquid culture medium, kanapenicillin with a final concentration of 45 to 55 μg / mL is added, and cultured at 35 to 40° C., 180 to 220 r / min for 10 to 15 hours, and then transferred to fresh LB liquid culture medium, kanapenicillin with a final concentration of 45 to 55 μg / mL is added, and cultured at 35 to 40° C., 180 to 220 r / min for 0.8 to 1.5 hours.

5. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 4, characterized in that: The buffer is Lysis buffer.

6. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 5, characterized in that: When the recombinant engineered bacteria are transferred to fresh LB liquid culture medium, lactose is added for induction after culturing for 1 hour.

7. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 6, characterized in that: The final concentration of lactose was 1 mM.

8. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 7, characterized in that: The lactose induction temperature was 20°C.

9. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 8, characterized in that: The lactose induction time was 20 h.

10. The method for producing human acetaldehyde dehydrogenase 2 by combining fusion expression and lactose induction according to claim 9, characterized in that: The specific steps for purifying NusA-ALDH2 using nickel column affinity chromatography are as follows: (1) Nickel column equilibration: Add 5 column volumes of Lysis buffer to the nickel column and allow gravity to flow out to equilibrate the column; (2) Loading: Add the crude NusA-ALDH2 enzyme solution to the equilibrated nickel column and let it flow out by gravity; (3) Washing: Add 10 column volumes of washing buffer to the nickel column and let gravity flow out to wash out the impurities bound to the nickel column; (4) Elution: Add 2 times the column volume of elution buffer to the nickel column and let gravity flow out. At this time, the target protein is displaced and eluted, and this part of the eluate is collected; (5) Dialysis: The eluate was transferred to a 14 kD dialysis bag, which was placed in a 20 mM Tris-HCl buffer and dialyzed overnight to reduce the imidazole concentration in the enzyme solution.

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