A method for preparing SUMO-tagged thiamine pyrokinase using a fermenter

Abstract

This invention discloses a method for preparing SUMO-tagged thiamine pyrophosphate kinase using a fermenter, belonging to the field of bioengineering technology. The method involves culturing glycerol bacteria containing recombinant genetically engineered bacteria in a 5L fermenter with 3L of culture medium and added oxygen carrier. The aeration rate is controlled at 3.5L / min and the rotation speed at 450 rpm. Isopropyl-β-D-thiogalactoside is added to a final concentration of 0.2 mM, and induction is performed at 20°C for 20 h. The target product is obtained after cell collection, resuspension, disruption, and centrifugation purification. This invention optimizes the fermenter process parameters, improving the soluble expression level of the target protein and the efficiency of large-scale preparation. The process is stable, reproducible, and suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for preparing SUMO-tagged thiamine pyrophosphate kinase using a fermenter. Background Technology

[0002] Thiamine pyrophosphate kinase (TPK) is a key enzyme in the metabolism of vitamin B1, specifically catalyzing the phosphorylation of thiamine (vitamin B1) to its active form, thiamine pyrophosphate (TPP). As an important coenzyme, TPP is widely involved in physiological processes such as carbohydrate metabolism and the tricarboxylic acid cycle. Therefore, TPK has broad application prospects in biocatalysis, pharmaceutical research and development, food additives, and clinical diagnostics.

[0003] Currently, there are significant technical challenges in the recombinant fermentation preparation of SUMO-TPK (SUMO-tagged thiamine pyrophosphate kinase): existing fermentation processes have not optimized fermenter parameters for the metabolic characteristics of SUMO-TPK, resulting in problems such as low parameter matching, unbalanced nutrient supply, unreasonable induction conditions, and poor dissolved oxygen control. This leads to uneven cell growth, unstable SUMO-TPK expression, low activity, and high inclusion body formation rate, making it difficult to achieve efficient and large-scale preparation.

[0004] As the core equipment for large-scale bio-fermentation, the fermenter's parameters (stirring speed, aeration rate, temperature, pH, dissolved oxygen, etc.) directly affect cell growth and target protein expression. Optimizing fermenter process parameters is key to improving SUMO-TPK preparation efficiency and achieving large-scale production. However, to date, a dedicated process for the large-scale preparation of SUMO-TPK using fermenters is not yet perfected. There is a lack of fermenter parameter combinations and nutrient supply strategies that precisely match the metabolic characteristics of SUMO-TPK, failing to fully leverage the advantages of large-scale and precise parameter control in fermenters and limiting the industrial application of SUMO-TPK.

[0005] Therefore, developing an efficient fermentation preparation method based on a fermenter, and optimizing fermenter operating parameters, nutrient supply, and induction conditions to solve the technical pain points of unstable SUMO-TPK expression, low activity, and difficulty in large-scale preparation, and to achieve efficient, stable, and large-scale production of SUMO-TPK, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] To overcome the above-mentioned technical defects, this invention provides a method for preparing SUMO-tagged thiamine pyrophosphate kinase using a fermenter. This method addresses the technical problems in the existing fermentation preparation of SUMO-tagged thiamine pyrophosphate kinase, which suffer from low fermenter parameter matching, unreasonable nutrient supply and induction conditions, and poor dissolved oxygen control. These problems result in uneven cell growth, unstable target protein expression, low activity, and high inclusion body formation rate. Furthermore, the lack of a fermenter-based large-scale preparation process adapted to its metabolic characteristics makes it difficult to achieve efficient large-scale production, thus limiting its industrial application.

[0007] This invention provides a method for preparing SUMO-tagged thiamine pyrokinase using a fermenter, comprising the following steps: (1) Take glycerol bacteria containing recombinant genetically engineered bacteria and culture them in a 5L fermenter. The fermenter contains 3L of liquid. Add oxygen carrier to the fermentation medium. During the culture, control the aeration rate at 3.5L / min and the rotation speed at 450r / min. Add isopropyl-β-D-thiogalactoside to a final concentration of 0.2mM and induce culture at 20℃ for 20h. (2) Centrifuge the induced fermentation broth in the fermenter at 4℃ and 4000rpm for 20 minutes, discard the supernatant, and collect the cells; (3) Add 200ml of 50mM Tris-HCl buffer solution to the collected bacterial cells and mix well. Use a cell disruptor to disrupt the bacterial cells. The disruption parameters are: disruption for 5 seconds, pause for 3 seconds, disruption power of 20%, and total disruption time of 30 minutes to obtain the bacterial cell disruption solution. (4) Centrifuge the bacterial cell lysate at 4°C and 12,000 rpm for 20 minutes and collect the supernatant; obtain SUMO-tagged thiamine pyrophosphate kinase by Ni-NTA affinity chromatography.

[0008] Furthermore, the recombinant genetically engineered bacteria was constructed by transforming the pET28a-SUMO-TPK expression vector into the BL21(DE3) strain.

[0009] Furthermore, the fermentation medium is TB medium, and the components and concentrations of the medium are: yeast extract 24g / L, tryptone 12g / L, glycerol 4mL, potassium dihydrogen phosphate 2.3g / L, and dipotassium hydrogen phosphate 16.4g / L.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses a 5L fermenter as the core to prepare SUMO-TPK. By optimizing the process parameters of the entire fermenter process, a special fermentation process adapted to the metabolic characteristics of SUMO-TPK is formed. This solves the technical pain points of low matching degree of process parameters and inaccurate parameter control in existing fermenters. It fully leverages the core advantages of fermenters in terms of large-scale production and precise temperature and parameter control, realizing the efficient and large-scale preparation of SUMO-TPK and breaking the limitations of existing fermentation processes on the industrial production of SUMO-TPK.

[0011] 2. By precisely optimizing key parameters of the fermenter, the oxygen transfer efficiency of the fermentation system is significantly improved, which fully meets the oxygen demand for the growth of E. coli and the expression of SUMO-TPK. This effectively avoids problems such as slow cell growth, protein misfolding, and increased inclusion bodies caused by insufficient dissolved oxygen, thereby improving the expression level and activity of SUMO-TPK.

[0012] 3. Optimize the nutrient supply and induction strategy of the fermenter, and adopt the integrated fermenter culture mode of "seed culture-scale culture", which eliminates the need for additional flask shaking operation and simplifies the process.

[0013] 4. The online monitoring and automatic adjustment function of the fermenter can ensure the stability of parameters such as temperature, pH, and dissolved oxygen during the fermentation process, effectively avoid the fluctuation of expression levels caused by human operation errors, improve process stability and experimental repeatability, and provide reliable process support for the large-scale industrial production of SUMO-TPK. At the same time, the integrated fermenter process simplifies operation steps, reduces labor costs and production energy consumption, and significantly improves production efficiency. Attached Figure Description

[0014] Figure 1 This is the SDS-PAGE electrophoresis pattern of SUMO-TPK obtained in Example 1. M is the protein molecular weight standard marker, 1 is the TPK-SUMO supernatant, 2 is the TPK-SUMO precipitate, 3 is the 250mM imidazole elution 1, 4 is the 250mM imidazole elution 2, 5 is the 250mM imidazole elution 3, and 6 is the flow-through buffer. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0016] Example

[0017] TB culture medium is prepared using a two-step method. The specific preparation steps are as follows (for preparing 1L of TB culture medium): 1. Preparation of TB-A solution: Weigh 24g of yeast extract, 12g of tryptone, and 4g of glycerol, dissolve them in 900mL of ultrapure water, stir until completely dissolved, autoclave at 121℃ for 20 minutes, and cool to room temperature for later use; 2. Preparation of TB-B solution: Weigh 2.3g of potassium dihydrogen phosphate and 16.4g of dipotassium hydrogen phosphate, dissolve them in 100mL of ultrapure water, stir until completely dissolved, autoclave at 121℃ for 15min, and cool to room temperature for later use. 3. Mix and adjust volume: Gently mix 900 mL of cooled TB-A solution with 100 mL of TB-B solution to obtain 1 L of complete TB culture medium.

[0018] High-efficiency fermentation preparation of SUMO-TPK in a fermenter: Glycerol-containing recombinant genetically engineered bacteria expressing SUMO-TPK were directly inoculated into a 5L fermenter for cultivation. The initial volume of the fermenter was 3L. An oxygen carrier was added to the fermentation medium to improve the oxygen transfer efficiency of the fermentation system. During cultivation, the aeration rate was controlled at 3.5L / min, the stirring speed at 450r / min, and the temperature, pH, and dissolved oxygen concentration of the fermentation system were maintained. After 8 hours of fermentation, IPTG inducer was added to the fermenter, with the final concentration controlled at 0.2mM. The fermenter temperature was adjusted to 20℃, and the above aeration rate and stirring speed were maintained for 20 hours to induce expression, obtaining the induced fermentation broth. Subsequently, the bacterial cells were collected. The induced fermentation broth was centrifuged at 4℃ and 4000rpm for 20 minutes, the supernatant was discarded, and the bacterial cell precipitate was collected. 200ml of 50mM IPTG was added to the collected bacterial cell precipitate. After thoroughly resuspending the bacterial cells in Tris-HCl buffer, the cells were lysed using a cell disruptor with the following parameters: 5 seconds of disruption followed by a 3-second pause, 20% disruption power, and a total disruption time of 30 minutes. The resulting bacterial lysate was centrifuged at 4°C and 12,000 rpm for 20 minutes. The supernatant was collected, and the crude enzyme solution was purified by Ni-NTA affinity chromatography to obtain high-purity SUMO-TPK. Protein expression was detected by SDS-PAGE electrophoresis.

[0019] Purification of SUMO-TPK: First, the Ni-NTA affinity chromatography column was equilibrated using a equilibration buffer consisting of 50 mM Tris-HCl buffer and 150 mM sodium chloride, with a volume of 25 mL. After equilibration, the supernatant was loaded onto the equilibrated Ni-NTA affinity chromatography column. After complete sample adsorption, contaminating proteins were eluted using a washing buffer consisting of 50 mM Tris-HCl buffer, 150 mM sodium chloride, and 20 mM imidazole, with a volume of 150 mL. After elution of contaminating proteins, the target protein was eluted using an elution buffer consisting of 50 mM Tris-HCl buffer, 150 mM sodium chloride, and 250 mM imidazole, with a volume of 30 mL. The elution peaks were collected. The collected fractions were then analyzed by SDS-PAGE electrophoresis.

Claims

1. A method for preparing SUMO-tagged thiamine pyrophosphate kinase using a fermenter, characterized in that, Includes the following steps: (1) Take glycerol bacteria containing recombinant genetically engineered bacteria and culture them in a 5L fermenter. The fermenter contains 3L of liquid. Add oxygen carrier to the fermentation medium. During the culture, control the aeration rate at 3.5L / min and the rotation speed at 450r / min. Add isopropyl-β-D-thiogalactoside to a final concentration of 0.2mM and induce culture at 20℃ for 20h. (2) Centrifuge the induced fermentation broth in the fermenter at 4℃ and 4000rpm for 20 minutes, discard the supernatant, and collect the cells; (3) Add 200ml of 50mM Tris-HCl buffer solution to the collected bacterial cells and mix well. Use a cell disruptor to disrupt the bacterial cells. The disruption parameters are: disruption for 5 seconds, pause for 3 seconds, disruption power of 20%, and total disruption time of 30 minutes to obtain the bacterial cell disruption solution. (4) Centrifuge the bacterial cell lysate at 4°C and 12,000 rpm for 20 minutes and collect the supernatant; obtain SUMO-tagged thiamine pyrophosphate kinase by Ni-NTA affinity chromatography.

2. The method according to claim 1, characterized in that, The recombinant genetically engineered bacteria were constructed by transforming the pET28a-SUMO-TPK expression vector into the BL21(DE3) strain.

3. The method according to claim 1, characterized in that, The fermentation medium is TB medium, and the components and concentrations of the medium are: yeast extract 24g / L, tryptone 12g / L, glycerol 4mL, potassium dihydrogen phosphate 2.3g / L, and dipotassium hydrogen phosphate 16.4g / L.

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