Expression and purification method of cyanobacteria malate dehydrogenase
By expressing and purifying cyanobacterial malate dehydrogenase in E. coli, the detailed research problem of cyanobacterial MDH was solved, and the new characteristics and application potential of cyanobacterial MDH were discovered, laying the foundation for its structural and functional research.
Patent Information
- Application Number
- CN202510085286.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-05-23
AI Technical Summary
It is difficult to study the biochemical properties, enzymatic properties, catalytic functions and coenzyme specificity of cyanobacterial malate dehydrogenase in detail, and the study on the enzymatic, structural and molecular evolution mechanisms of the cyanobacterial MDH family is relatively insufficient.
A method for expression and purification of cyanobacterial malate dehydrogenase is proposed, including transforming the constructed recombinant plasmid into E. coli cells, inoculating bacterial fluid, fermenting and culture, collecting bacteria and purifying by cell fragmentation and chromatography to obtain the purified cyanobacterial malate dehydrogenase protein.
Through this method, the foundation for the structural and functional research of cyanobacteria MDH was laid, and it was found that the cyanobacteria MDH family may have new characteristics of bicoenzyme dependence, have good heat resistance and thermal stability, and have good industrial application potential.
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Figure CN120025995A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of enzyme engineering, and in particular to an expression and purification method of cyanobacterial malate dehydrogenase. Background Art
[0002] Malate dehydrogenase (MDH) is widely distributed, from the cytoplasm to various organelles, from prokaryotes to true bacteria, archaea and higher plants and animals. MDH catalyzes the reversible conversion between malate and oxaloacetate, plays an important role in the central metabolism of cells such as the tricarboxylic acid cycle and the shuttling of malate and aspartate, and is also involved in the stress response caused by various environmental stresses in plants.
[0003] Cyanobacteria, also known as blue-green algae, are Gram-negative single-cell prokaryotes. As the earliest photosynthetic autotrophic organisms on Earth, the scientific classification of different species of Cyanobacteria helps us understand the evolutionary relationship between different groups of organisms, and is of great significance for us to trace the origin of life and explore the evolutionary process of organisms.
[0004] The incompleteness of the TCA cycle is often used to explain the current different views on the physiological metabolism of cyanobacteria. There have been many studies on the basic metabolism of cyanobacteria, but the research on the molecular evolutionary mechanisms of these major metabolic enzymes is relatively insufficient, among which the research on the enzymology, structure and molecular evolutionary mechanism of the cyanobacterial MDH family is particularly poor.
[0005] Currently, there are few studies on heterologous expression and purification of MDH gene in E. coli, which makes it difficult to conduct detailed studies on its biochemical properties, enzymatic properties, catalytic function and coenzyme specificity. Summary of the invention
[0006] In view of this, the purpose of the present invention is to provide a method for expressing and purifying cyanobacterial malate dehydrogenase, so as to facilitate detailed research on its biochemical properties, enzymatic properties, catalytic function and coenzyme specificity.
[0007] Based on the above purpose, the present invention provides a method for expressing and purifying cyanobacterial malate dehydrogenase, comprising the following steps:
[0008] S1. Transform the constructed recombinant plasmid into competent E. coli cells;
[0009] S2, inoculating and fermenting the bacterial solution obtained in S1, and collecting the bacterial cells;
[0010] S3, adding the bacterial cells into a buffer to disrupt the cells to obtain a supernatant;
[0011] S4. Add the supernatant to resin, and obtain purified cyanobacterial malate dehydrogenase protein through chromatography and elution.
[0012] As an implementation method, taking Chromococcus as an example, the construction method of the recombinant plasmid pApFPU1MDH is as follows:
[0013] According to the gene sequence of Aphanothece sacrum FPU1 MDH in GenBank, the gene fragment required in the experiment was synthesized. The MDH gene (synthesized by General Biosystems (Anhui) Co., Ltd.) and the vector pET-28b (+) were double-digested with Nde I and Xho I DNA restriction endonucleases at the same time. The digested fragments were cloned by T 4 -DNA ligase ligation, the ligation product was transformed into E.coil DH5α host cells, and positive transformation clones were selected after kanamycin resistance screening.
[0014] Extraction and identification of recombinant plasmid
[0015] The positive clones obtained in the above steps were expanded and cultured, and plasmid DNA was extracted using a kit. The method is shown in the instruction manual of the "High Purity Plasmid Small-Amount Rapid Extraction Kit HiPu Plasmid Mini Kit". The obtained plasmid was preliminarily detected and identified by agarose gel electrophoresis and double restriction digestion with Nde I and Xho I, and DNA sequencing was used to identify whether the pApFPU1MDH plasmid was successfully recombined. The recombinant plasmid was identified by agarose gel electrophoresis.
[0016] The amino acid sequence of the above-mentioned Aphanothece sacrum FPU1 MDH is as follows:
[0017]
[0018]
[0019] The nucleotide sequence of the Aphanothece sacrum FPU1 MDH gene is as follows:
[0020] The method for transforming the constructed recombinant plasmid into Escherichia coli competent cells comprises the following steps:
[0021] (1) Thaw the competent E. coli cells and pCyMDH recombinant plasmid on ice;
[0022] (2) Mix the recombinant plasmid and competent cells and place on ice for 25-35 minutes;
[0023] (3) After heat shock at 42°C for 90 seconds, the cells were immediately placed on ice for 5 minutes, and LB medium without resistance was added, followed by incubation at 37°C and 225 rpm for 1 hour.
[0024] (4) After centrifugation, remove the supernatant and resuspend the cells to obtain bacterial solution;
[0025] (5) Spread the bacterial solution evenly on LB solid medium and culture at 37°C overnight.
[0026] The method of bacterial liquid inoculation and fermentation culture in S2 comprises the following steps:
[0027] (1) Pick a single colony and inoculate it into LB medium and culture it at 37°C and 225 rpm overnight;
[0028] (2) Take the overnight culture solution and inoculate it into LB medium, culture it at 37°C and 225 rpm until the OD600 is 0.4-0.6, add 1 M IPTG to a final concentration of 0.5 mM, and culture it at 20°C and 180 rpm for 20 h;
[0029] (3) Place the bacterial solution on ice, centrifuge, and collect the cells.
[0030] The method for cell disruption in S3 comprises the following steps:
[0031] (1) Add pre-cooled buffer to the cells, shake thoroughly to suspend the cells, and discard the supernatant after centrifugation to remove the residual culture medium;
[0032] (2) Add buffer again, shake thoroughly to suspend the precipitate, place the centrifuge tube in an ice-water mixture, and disrupt the cells by ultrasonication;
[0033] (3) The disrupted liquid was centrifuged at 12,000 rpm for 20 min at 4°C, the precipitate was discarded, and the supernatant was retained.
[0034] S4 The method for purifying cyanobacterial malate dehydrogenase protein comprises the following steps:
[0035] (1) Equilibrate Co with buffer 2+ Resin, add the supernatant to the resin, and flip at 4°C to allow the protein to fully bind to the resin;
[0036] (2) Load the protein-bound resin into a chromatography column and wash the column with 5 column volumes of buffer;
[0037] (3) Elute the target protein with elution buffer containing 150 mM imidazole, collect and aliquot the eluate into centrifuge tubes, and store on ice or at 4°C for later use.
[0038] Beneficial effects of the present invention: The present invention lays a foundation for the study of the structure and function of cyanobacterial MDH. Studies have shown that the cyanobacterial MDH family may have a new feature of dual coenzyme dependence, and MDH from some species of cyanobacteria has good heat resistance and thermal stability, and has good industrial application potential. In addition, studies have found that cyanobacterial MDH exists in a tetrameric form that is more similar to LDH, which not only increases the complexity of the cyanobacterial MDH family, but also provides a new path for studying the systematic evolution of cyanobacterial MDH. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0040] Figure 1 Identification of the pApFPU1MDH recombinant plasmid of the present invention; A, M: DL5000 bp DNA marker; 1: pET28b(+); 2: pApFPU1MDH; B, M: DL10 kbp DNA marker; 1: Double digestion product of pApFPU1MDH;
[0041] Figure 2 The SDS-PAGE detection of CyMDH of the present invention; M: Protein Marker; 1-7. Nostocales MDH: Ce953MDH, No9414MDH, Ca3974MDH, Sc4073MDH, Sc61278MDH, Ns1411MDH, No25MDH; 8-13. Oscillatoriales MDH: PhOSCRMDH, Mi7113MDH, Le15MDH, Le6406MDH, Ge7105MDH, Ar9108MDH, Ly8106MDH; 14-18. Chromococcus MDH: ApFPU1MDH, Ap016MDH, Gl7822MDH, Gl7424MDH;
[0042] Figure 3 This is the gel filtration chromatography analysis of CyMDH of the present invention; A: Chromococcales ApFPU1MDH; B: Nostocales Sc4073MDH; C: Oscillatorales Le15MDH;
[0043] Figure 4 This is the gel filtration chromatography analysis of CyMDH after desalting treatment of the present invention; A: ApFPU1MDH; B: Sc4073MDH. DETAILED DESCRIPTION
[0044] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.
[0045] The experimental materials used in the present invention are as follows:
[0046] 1. Strains and plasmids
[0047]
[0048] Main reagents: Isopropyl-β-D-thiogalactoside (IPTG), oxaloacetic acid (OAA), NDAH, and NADPH were purchased from Sigma-Aldrich; Tris, EDTA, and antibiotics were purchased from Sangon Biotechnologies (Shanghai) Co., Ltd.; and protein molecular weight standard (ProMantTM Prestained protein Ladder) was purchased from Beijing Protein Biotechnologies Co., Ltd.
[0049] The high-purity plasmid small-scale rapid extraction kit was purchased from Protein Biotechnologies; the modified Bradford Protein Assay Kit was purchased from Shanghai Industrial Biotechnology Co., Ltd. and Co 2+ Affinity chromatography resin was purchased from TaKaRa Co., Ltd. Other conventional reagents were kept in the laboratory.
[0050] Liquid LB medium (1L):
[0051]
[0052] Use ddH 2 After O is completely dissolved, the volume is adjusted to 1L, and the required amount is divided and then sterilized by high-pressure steam (121°C, 20min). According to the experimental needs, the corresponding antibiotics are added. The final concentrations of kanamycin (Kanamycin, Kan) and chloramphenicol (Chloramphenicol, Cam) are 30μg / ml and 15μg / ml, respectively.
[0053] Antibiotic solution (30 mg / ml):
[0054] Antibiotics Kan and Cam: weigh 300 mg of antibiotics and dissolve in 10 ml ddH 2 O, sterilize with a 0.22 μm filter membrane under sterile conditions, aliquot under sterile conditions, and store at -20°C for later use.
[0055] Lysis / Equilibrium / Washing Buffer(LEW Buffer)(1L)
[0056] Ingredients and Dosages
[0057] NaH 2 PO 4 7.8g
[0058] NaCl 17.54g
[0059] The above ingredients were mixed in ddH 2 After the solution is completely dissolved in 4% O, adjust the pH to 8.0 with NaOH, make the volume to 1 L, and store at 4°C for later use.
[0060] Elution Buffer(150mM,500ml):
[0061]
[0062] Dissolve the above ingredients in 400 ml ddH 2 O water, adjust the pH to 8.0 with NaOH and make up to 500 mL.
[0063] Elution Buffer (20mM, 500mL):
[0064]
[0065] Dissolve the above reagents in 400 ml ddH 2 O water, adjust the pH to 8.0 with NaOH and make up to 500 mL.
[0066] Polyacrylamide gel
[0067]
[0068] The 30% acrylamide solution was prepared from NN-methylenebisacrylamide and acrylamide, and was stored at 4°C in the dark after filtration.
[0069] Protein loading buffer (200ml)
[0070]
[0071] DTT was added proportionally to a final concentration of 100 mM before use.
[0072] 5× Protein Electrophoresis Buffer (1L)
[0073]
[0074]
[0075] Use ddH 2 O to 1L, dilute to 1× before use.
[0076] Coomassie Brilliant Blue Staining Solution (100ml)
[0077]
[0078] Destaining solution (100ml)
[0079]
[0080] Preparation of competent E. coli
[0081] The bacteria stored at -80°C were streaked on plates to activate the strains and incubated upside down at 37°C overnight;
[0082] Pick a single colony of the sample clone, inoculate it into 5 ml of LB medium containing the corresponding antibiotics, and culture it at 37°C and 225 rpm overnight;
[0083] Take 500 μl of overnight cultured bacteria and inoculate into 50 ml of LB medium containing corresponding antibiotics, and culture at 37°C and 225 rpm;
[0084] Wait for the bacterial solution OD 600 When the pH value is 0.3-0.5, transfer it to a pre-cooled 50 ml centrifuge tube and place it in an ice bath for 10 min.
[0085] Centrifuge at 4°C, 5000 rpm for 10 min and discard the supernatant;
[0086] Add 15 ml of 0.1 M pre-cooled CaCl 2 , after suspending the precipitate, place in ice bath for 30 min;
[0087] Centrifuge at 4°C, 5000 rpm for 10 min and discard the supernatant;
[0088] Add 3 ml of 0.1 M pre-cooled CaCl 2 0% glycerol, resuspended pellet;
[0089] Use EP tubes to divide into 100 μl portions and store at -80°C for later use.
[0090] Recombinant plasmid construction and identification
[0091] The cyanobacterial strains and MDH genes selected in this study are shown in the table. According to the gene sequence of MDH in GenBank, all genes were synthesized by General Biosystems (Anhui) Co., Ltd. The gene fragment and vector pET28b (+) were double-digested with restriction endonucleases (Nde I and Xho I) and then cloned with T 4 -DNA ligase is used for ligation, and the ligation product is transformed into E. coli DH5α competent cells. Positive transformants are selected for expansion and culture, and the recombinant plasmid is extracted for DNA sequencing identification.
[0092]
[0093] Transformation of recombinant plasmid into E.coli Rosetta (DE3) competent cells
[0094] Thaw the DE3 competent cells and pCyMDH recombinant plasmid on ice for 10 min;
[0095] Take 2 μl of recombinant plasmid and mix with competent cells, and place on ice for 30 min;
[0096] After heat shock at 42°C for 90 seconds, the cells were immediately placed on ice for 5 minutes, 500 μl of resistance-free LB medium was added, and the cells were cultured at 37°C and 225 rpm for 1 hour;
[0097] After centrifugation at 4000 rpm for 1 min, 400 ml of supernatant was removed and the cells were resuspended;
[0098] Take 100 μl of the above bacterial solution and spread it evenly on LB solid medium containing Kan and Cam resistance, and culture it at 37°C overnight.
[0099] Inducible expression of cyanobacterial MDH protein
[0100] Pick a single colony from the plate in 1.2.1.3 and inoculate it into 5 ml of LB medium containing Kan and Cam resistance, and culture it at 37°C and 225 rpm overnight;
[0101] Take 3 ml of overnight cultured bacteria and inoculate into 200 ml LB medium containing Kan and Cam resistance, and culture at 37°C and 225 rpm until OD 600 0.4-0.6, add 1M IPTG to a final concentration of 0.5mM, and culture at 20°C and 180rpm for 20h;
[0102] The bacterial solution was placed in an ice bath for 5 min and centrifuged at 5000 rpm for 5 min to collect the bacteria.
[0103] Purification and identification of cyanobacterial MDH protein
[0104] Cell disruption
[0105] Add 25 ml of pre-cooled LEW to the cells, shake thoroughly to suspend the cells, centrifuge at 4°C, 5000 rpm, and discard the supernatant to remove the residual culture medium;
[0106] Add 25 ml of LEW again, shake thoroughly to suspend the precipitate, place the centrifuge tube in an ice-water mixture, and disrupt the cells with ultrasound (ultrasound conditions: power 200 W, disruption 1 s, rest 2 s, total ultrasound time 40 min);
[0107] The broken liquid was centrifuged at 4°C, 12000rpm for 20min, the precipitate was discarded and the supernatant was retained.
[0108] Protein purification
[0109] Balancing Co with LEW 2+ Resin: Add the supernatant from the previous step to the resin and place it on a vertical mixer for 30 minutes at 4°C to allow the protein to fully bind to the resin;
[0110] Load the protein-bound resin into the chromatography column and wash the column with 5 column volumes of LEW buffer;
[0111] If necessary, the column can be washed with LEW buffer containing a gradient of low imidazole concentrations to remove non-specific proteins;
[0112] Elute the target protein with 5 ml of Elution buffer containing 150 mM imidazole, collect and dispense the eluate into centrifuge tubes, and store on ice or at 4°C for later use.
[0113] SDS-PAGE detection
[0114] Prepare 12% separation gel, inject it into the interlayer of the installed rubber plate so that the water level is 2 cm below the top of the rubber plate, and slowly add pure water for liquid sealing;
[0115] Let it stand at 37℃ for about 20 minutes, and remove as much pure water as possible after the separation gel solidifies;
[0116] Prepare 5% concentrated gel, fill the gel plate with it, insert the comb, and let it stand at room temperature until it solidifies;
[0117] Carefully pull out the comb from the gel plate, assemble the gel plate and the vertical electrophoresis tank, and add an appropriate amount of 1× Tris-Gly electrophoresis buffer;
[0118] Mix the loading buffer and sample protein in a 1:1 ratio, boil in a boiling water bath for 5 minutes, centrifuge briefly to collect the sample, and add an appropriate amount of sample to the loading well of the gel;
[0119] Electrophoresis was performed at a constant voltage of 160 V until the bromophenol blue indicator reached the bottom of the separation gel;
[0120] Take out the separation gel, place it in Coomassie Brilliant Blue staining solution, and stain it fully on a horizontal shaker;
[0121] The separation gel was destained in a destaining solution until the gel background was transparent and the protein bands were clear, and photographed for record.
[0122] Gel filtration chromatography
[0123] Prepare LEW and ddH in advance 2 O and 20% ethanol, and filter before use;
[0124] Turn on AKTA Protein Purifier and use ddH 2 O Flush the pump first and then flush the Superdex increase20010 / 300Column, and set the flow rate to 0.5ml / min and the maximum alarm pressure to 3.0MPa;
[0125] Repeat the previous step with LEW;
[0126] Rinse the sample loop with LEW, add 500 μl of pretreated (concentrated, degassed) protein sample with a syringe, start filtration chromatography, and collect data;
[0127] After chromatography, ddH 2 Rinse the pump and column with 3% O and 20% ethanol.
[0128] Protein concentration determination
[0129] All protein concentrations in this experiment were determined using the Modified Bradford Protein Assay Kit. According to the instructions, a protein standard curve was first prepared:
[0130] Dilute the BSA standard protein to different concentrations with distilled water: 50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml, 300 μg / ml;
[0131] Take 100 μl of standard protein of different concentrations and add them into different EP tubes. The control group uses 100 μl of ddH 2 O instead of the standard band, then add 1 ml of Bradford reagent to each tube and shake thoroughly to mix;
[0132] After standing at room temperature at 25-30°C for 10 minutes, the A of standard proteins with different concentrations was detected. 595 Light absorption value;
[0133] The protein concentration is used as the horizontal axis, corresponding to the A of each tube. 595 The value is the ordinate, and the standard curve is drawn;
[0134] Sample concentration determination: Mix 100 μl sample protein with 1 ml Bradford reagent and let stand at room temperature for 10 min before testing its A 595 The sample concentration is calculated based on the standard curve. (This method is only applicable to samples with a concentration of <300μg / ml. If the concentration is greater than 300μg / ml, the protein needs to be diluted using LEW for accurate determination.)
[0135] Identification of pCyMDH recombinant plasmid
[0136] The ligation product of the target gene MDH and the vector pET28b(+) was transformed into E.coil DH5α, and the positive clones were expanded to extract the plasmids, and then detected by gel electrophoresis and double restriction digestion with Xho I and Nde I. The results were ( Figure 1 ) shows that the size of the recombinant plasmid is consistent with the expectation, and after double enzyme digestion, a fragment of the target gene size is generated, indicating that the recombinant plasmid is successfully constructed. The construction and identification methods of the recombinant plasmid are the same, and only the detection result picture of pApFPU1MDH is listed in this article.
[0137] Expression and purification of cyanobacterial MDH
[0138] SDS-PAGE of CyMDH
[0139] The recombinant plasmid was transformed into E. coli Rosetta (DE3) competent cells. After IPTG induction of target gene expression, the cyanobacterial MDH genes selected in the experiment could be expressed in soluble form in E. coli. 2+ The different cyanobacterial MDH proteins purified by affinity chromatography were detected by SDS-PAGE. Figure 2 As shown, the molecular weight of MDH is between 35-40 kDa, and the purity can meet the requirements of subsequent enzymatic experiments.
[0140] Gel filtration chromatography
[0141] Gel filtration chromatography was performed on one MDH from each of the three orders of cyanobacteria: ApFPU1MDH from Chromococcus, Sc4073MDH from Nostoc, and Le15MDH from Oscillatoria, to analyze the polymerization form and molecular weight of the CyMDH holoenzyme. The results are shown in the figure. Figure 3: As can be seen from the figure, the gel filtration chromatography results of ApFPU1MDH, Sc4073MDH and Le15MDH all have only a single peak, and appear at elution volumes of 12.83ml, 12.87ml and 13.1ml respectively. Referring to the standard protein elution curve, the molecular weight of the whole enzyme of MDH is calculated to be 139.5kDa, 136.9kDa and 122.7kDa respectively, and the molecular weight of a single subunit of cyanobacterial MDH is about 34.5kDa. The results preliminarily indicate that ApFPU1MDH, Sc4073MDH and Le15MDH are homotetramers.
[0142] To further exclude the possibility that the tetramer results were caused by imidazole in the eluate, the eluate containing the purified protein was desalted for gel filtration chromatography analysis. The results are shown in Figure 4 As shown in the figure: After desalting, ApFPU1MDH and Sc4073MDH have a main peak at the elution volume of 12.75ml, and a small peak appears at the elution volume of about 21.4ml. The position of the main peak is basically consistent with the position of the main peak in the result without desalting; through calculation, the small peak at about 21.4ml may be caused by impurities, indicating that the oligomeric state of the tetramer of cyanobacterial MDH exists naturally, not caused by imidazole.
[0143] Those skilled in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present invention is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other changes in different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity. Any omissions, modifications, equivalent substitutions, improvements, etc. 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 expressing and purifying cyanobacterial malate dehydrogenase, characterized in that: The steps include: S1. Transform the constructed recombinant plasmid into competent E. coli cells; S2, inoculating and fermenting the bacterial solution obtained in S1, and collecting the bacterial cells; S3, adding the bacterial cells into a buffer to disrupt the cells to obtain a supernatant; S4. Add the supernatant to resin, and obtain purified cyanobacterial malate dehydrogenase protein through chromatography and elution.
2. The method for expressing and purifying cyanobacterial malate dehydrogenase according to claim 1, characterized in that: The method for transforming the constructed recombinant plasmid into Escherichia coli competent cells comprises the following steps: (1) Thaw the competent E. coli cells and pCyMDH recombinant plasmid on ice; (2) Mix the recombinant plasmid and competent cells and place on ice for 25-35 minutes; (3) After heat shock at 42°C for 90 seconds, the cells were immediately placed on ice for 5 minutes, and LB medium without resistance was added, and cultured at 37°C and 225 rpm for 1 hour; (4) After centrifugation, remove the supernatant and resuspend the cells to obtain bacterial solution; (5) Spread the bacterial solution evenly on LB solid medium and culture at 37°C overnight.
3. The method for expressing and purifying cyanobacterial malate dehydrogenase according to claim 1, characterized in that: The method of bacterial liquid inoculation and fermentation culture in S2 comprises the following steps: (1) Pick a single colony and inoculate it into LB medium and culture it at 37°C and 225 rpm overnight; (2) Take the overnight culture solution and inoculate it into LB medium, culture it at 37°C and 225 rpm until the OD600 is 0.4-0.6, add 1 M IPTG to a final concentration of 0.5 mM, and culture it at 20°C and 180 rpm for 20 h; (3) Place the bacterial solution on ice, centrifuge, and collect the cells.
4. The method for expressing and purifying cyanobacterial malate dehydrogenase according to claim 1, characterized in that: The method for cell disruption in S3 comprises the following steps: (1) Add pre-cooled buffer to the cells, shake thoroughly to suspend the cells, and discard the supernatant after centrifugation to remove the residual culture medium; (2) Add buffer again, shake thoroughly to suspend the precipitate, place the centrifuge tube in an ice-water mixture, and disrupt the cells by ultrasonication; (3) The disrupted liquid was centrifuged at 12,000 rpm for 20 min at 4°C, the precipitate was discarded, and the supernatant was retained.
5. The method for expressing and purifying cyanobacterial malate dehydrogenase according to claim 1, characterized in that: S4 The method for purifying cyanobacterial malate dehydrogenase protein comprises the following steps: (1) Equilibrate Co with buffer 2+ Resin, add the supernatant to the resin, and flip at 4°C to allow the protein to fully bind to the resin; (2) Load the protein-bound resin into a chromatography column and wash the column with 5 column volumes of buffer; (3) Elute the target protein with elution buffer containing 150 mM imidazole, collect and aliquot the eluate into centrifuge tubes, and store on ice or at 4°C for later use.