Escherichia coli culture medium and application thereof in preparation of cell extract required by cell-free protein synthesis

Abstract

The invention discloses an Escherichia coli culture medium and application thereof in preparation of cell extracts required by cell-free protein synthesis, the culture medium comprises an initial culture medium or an initial culture medium and a supplementary culture medium, the initial culture medium comprises fructose, lactose, magnesium sulfate, ammonium sulfate, dipotassium phosphate, monopotassium phosphate, citric acid, trace elements, a vitamin solution and a nitrogen source; the fed-batch culture medium comprises a carbon source and a nitrogen source, the carbon source is fructose and a magnesium sulfate solution, and the nitrogen source comprises a nitrogen source solution added with yeast powder and yeast peptone. According to the culture medium disclosed by the invention, the escherichia coli can maintain logarithmic phase growth and reach relatively high thallus density (OD600 is greater than or equal to 60), and the prepared escherichia coli cell extract is used for cell-free protein synthesis, has high activity consistent with that of a cell extract obtained by low-density fermentation, and can be used for expressing various foreign proteins. According to the invention, the problem of low thallus density during preparation of the cell-free protein synthetic cell extract is solved, and the expression yield of the cell-free protein is effectively improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an Escherichia coli culture medium and application thereof in preparing a cell extract required for cell-free protein synthesis. Background Art

[0002] Cell-free protein synthesis technology is a new technology that produces proteins by transcription and translation in vitro without the participation of living cells. It has attracted more and more attention and application from researchers. Cell-free protein synthesis utilizes active ingredients in cell extracts, such as ribosomes, translation factors, aminoacyl-tRNA synthetases, enzymes related to energy metabolism, etc., and adds NTP, amino acids, energy substances, buffer salts, metal ions and other substances to synthesize target proteins in the presence of DNA templates or RNA templates. Compared with traditional recombinant expression technology, it has the advantages of being fast, flexible, high-throughput, and open, and can express toxic proteins, membrane proteins, non-natural amino acid embedded proteins and other proteins that are difficult to express by traditional means.

[0003] Whether it is scientific research reports or commercial kits, E. coli cell-free is the most involved, because E. coli grows fast, has high recombinant expression, perfect genetic modification, and is the most thoroughly researched. Many companies around the world have developed commercial cell-free protein expression kits, such as Suzhou Perrotin, NEB, Promega and other companies. However, the current E. coli cell-free protein synthesis system still has limitations. The biggest problem is that the reaction volume is still relatively small and the cost is relatively high. The most important reason for this limitation is the low efficiency of the preparation of cell extracts. The high-density fermentation of E. coli, which is usually used for intracellular recombinant expression, has a bacterial density (measured in absorbance OD 600 Calculation) can reach more than 50, and the solid content of the fermentation liquid can reach more than 10%. However, the E. coli fermentation used to prepare the cell extract required for cell-free protein synthesis must be harvested in the logarithmic growth phase to ensure that the cell extract has the best activity, resulting in the OD at the end of the general fermentation. 600 The value can only be around 1.5-3, which is more than 20 times different from the usual high-density fermentation. As a result, most cell extracts are obtained by shake flask fermentation, and in a few cases where fermentation is used, the collected OD 600 To expand the cell-free protein expression system to more industrial uses, it is necessary to expand its reaction system and reduce its raw material cost. 600 , is the key to achieving the above goals.

[0004] According to the literature (Zawada et al. Biotechnol Bioeng 94: 618-624), the activity of the cell extract is strictly related to the specific growth rate μ of Escherichia coli. The logarithmic growth period μ of Escherichia coli is greater than 1.0 h. -1 At this time, the metabolism of the bacteria is the most vigorous, and the ribosomes, tRNA and other translation-related factors contained in the bacteria reach the maximum abundance, which is conducive to the cell-free protein expression reaction. In the usual high-density fermentation, the specific growth rate μ is reduced to 0.3h -1 , the activity of the cell extract is also greatly reduced. In addition, when the bacteria are in the non-logarithmic growth phase, the content of transcription and translation inhibitors inside them increases accordingly, which is not conducive to cell-free protein expression. Therefore, by designing a new culture medium formula and adjusting and optimizing the feeding strategy, keeping the bacteria in the logarithmic growth phase and maintaining a large specific growth rate μ during the fermentation process is the key to increasing the harvest OD 600 The key.

[0005] Common culture medium formulas for E. coli include LB, 2YTP, 2YTPG, etc., which contain yeast extract, soy protein jelly, phosphate buffered saline and glucose. Glucose is a carbon source that can be commonly used by E. coli, which can be quickly utilized by E. coli and is conducive to its rapid growth. When glucose is used as a carbon source, it will produce a glucose effect, resulting in the accumulation of metabolic intermediate acetic acid. A document (Lee SY. Trends Biotechnol 14: 98-105) reported that when glucose is used as a carbon source, when the specific growth rate is greater than 0.3h -1 When the concentration of acetic acid is too high, it will accumulate acetic acid, thus affecting the specific growth rate and the final bacterial OD 600 In the batch-fed-batch culture process, the glucose feeding rate often needs to be carefully and complexly regulated to keep the residual sugar at a very low level to avoid the accumulation of acid. Therefore, from this perspective, glucose is not an ideal carbon source for the preparation of cell extracts, and it is very necessary to study the effects of other carbon sources on the preparation of cell extracts. Summary of the invention

[0006] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide an Escherichia coli culture medium, which solves the problem of low cell density when preparing Escherichia coli cell extracts required for cell-free protein synthesis, and effectively improves the yield of cell-free protein expression.

[0007] The present invention also provides an application of an Escherichia coli culture medium in preparing a cell extract, wherein the OD of the cell extract prepared on a 100L fermentation scale is 600 It increased from 1-3 to over 60, greatly improving the preparation efficiency of cell extracts and reducing costs.

[0008] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides an Escherichia coli culture medium, which includes an initial culture medium or an initial culture medium and a feed culture medium, wherein the initial culture medium includes fructose, lactose, magnesium sulfate, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, citric acid, trace elements and vitamin solution, and a nitrogen source, and the feed culture medium includes a carbon source and a nitrogen source, wherein the carbon source is fructose and a magnesium sulfate solution, and the nitrogen source in the initial culture medium and the feed culture medium is a nitrogen source solution to which yeast powder and yeast peptone are added.

[0009] Furthermore, the initial culture medium includes 1-3% fructose, 0.1-1% lactose, 0.1-0.5% magnesium sulfate, 0.1-0.4% ammonium sulfate, 0.5-5% dipotassium hydrogen phosphate, 0.2-3% potassium dihydrogen phosphate, 0.1-2% citric acid, 0.01%-0.5% trace element and vitamin solution, and 0.5-2% nitrogen source solution.

[0010] Preferably, the dosage of the initial culture medium is: 1-3% fructose, 0.5% lactose, 0.2% magnesium sulfate, 0.25% ammonium sulfate, 2% dipotassium hydrogen phosphate, 0.75% potassium dihydrogen phosphate, 0.22% citric acid, 0.1% trace element and vitamin solution, and 1% nitrogen source solution.

[0011] Furthermore, the trace element and vitamin solution includes copper sulfate pentahydrate, zinc chloride, ferrous sulfate heptahydrate, anhydrous cobalt chloride, calcium chloride dihydrate, manganese sulfate monohydrate, biotin, vitamin B12, and folic acid dihydrate.

[0012] Furthermore, the concentrations of the trace elements and vitamin solutions are: copper sulfate pentahydrate 0.1-2g / L, zinc chloride 0.1-0.7g / L, ferrous sulfate heptahydrate 1-5g / L, anhydrous cobalt chloride 0.2-4g / L, calcium chloride dihydrate 0.5-6g / L, manganese sulfate monohydrate 0.5-5g / L, biotin 0.05-0.2g / L, vitamin B12 0.005-0.02g / L, and folic acid dihydrate 0.01-0.2g / L.

[0013] Preferably, the concentrations of the trace element and vitamin solution are: copper sulfate pentahydrate 0.28 g / L, zinc chloride 0.14 g / L, ferrous sulfate heptahydrate 2.8 g / L, anhydrous cobalt chloride 1.3 g / L, calcium chloride dihydrate 1.5 g / L, manganese sulfate monohydrate 1.9 g / L, biotin 0.1 g / L, vitamin B12 0.01 g / L, and folic acid dihydrate 0.07 g / L.

[0014] Furthermore, the fructose concentration in the carbon source is 300-600 g / L, and the magnesium sulfate concentration is 10-20 g / L.

[0015] Furthermore, the concentration of yeast powder in the nitrogen source solution is 20-100 g / L, and the concentration of yeast peptone is 50-150 g / L.

[0016] The invention discloses an application of the Escherichia coli culture medium in preparing cell extracts.

[0017] The invention discloses an application of the Escherichia coli culture medium in preparing a cell extract required for cell-free protein synthesis.

[0018] Furthermore, the application process includes the following steps:

[0019] (1) Batch-fed-batch fermentation was carried out in a fermenter containing Escherichia coli at elevated temperature. When the carbon source in the initial culture medium was exhausted and the dissolved oxygen increased rapidly, carbon source and nitrogen source were added and the OD was measured. 600 , stop fermentation, centrifuge, resuspend and wash to collect the cells;

[0020] (2) Add the bacterial cells prepared in step (1) into S30 buffer solution, crush them, and centrifuge them to collect the supernatant, which is the cell extract.

[0021] Furthermore, in step (1), the amount of carbon source and nitrogen source added is 10-15 ml respectively, the carbon source addition rate is 0.2-0.45 ml / L / min, and the nitrogen source addition rate is 20-40% of the carbon source.

[0022] Preferably, the initial feeding rate of the carbon source in step (1) is 0.33 ml / L / min, and then the carbon source is fed at an exponential rate k=1.0 h -1 The nitrogen source feed rate was maintained at 30% of the carbon source.

[0023] Furthermore, in step (1), the OD 600 Is 1.5-3.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0025] The novel culture medium and optimized feeding strategy provided by the present invention can enable Escherichia coli to maintain logarithmic growth and a large specific growth rate during fermentation and culture; and can increase the OD value of the cell extract to 600 Increasing it to above 60 and maintaining the optimal activity greatly improves the preparation efficiency of cell extracts and reduces the cost of the cell-free protein expression system; it lays the foundation for the scale-up of the cell-free protein expression system and expands its application range.

[0026] For high-density fermentation, the present invention optimizes the feeding strategy during the fermentation process, so that the bacteria can maintain a large specific growth rate μ and remain in the logarithmic growth phase. Finally, the OD600 Up to 60% cell extracts can be used for cell-free protein expression to achieve the same protein yield as cell extracts harvested at low density. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the standard curve of sfGFP protein concentration and fluorescence value;

[0028] Figure 2 are the growth curves of different carbon sources;

[0029] Figure 3 is the OD at different exponential feeding rates 600 Change curve;

[0030] Figure 4 It is a comparison between 2YTPG medium and the medium and feeding strategy of this patent;

[0031] Figure 5 Comparison of the activity of the cell extracts prepared by 2YTPG medium and the present invention (from left to right: negative control, 2YTPG medium, the present invention);

[0032] Figure 6 This is the fermentation curve of 100L;

[0033] Figure 7 OD is the bacterial density 600 Plots of changes over time;

[0034] Figure 8 This is a diagram of the vector construction for DNase I;

[0035] Fig. 9 It is the SDS-PAGE electrophoresis of cell-free protein expressing DNase I;

[0036] Fig.10 It is a crude enzyme activity assay for cell-free protein expressed DNase I. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0039] Example 1

[0040] Establishment of a cell-free protein expression system (the method and system constructed in this example are used for subsequent initial culture medium and feed screening experiments)

[0041] (1) Preparation of cell extracts

[0042] Escherichia coli BL21 Star (DE3) strain was selected as the experimental strain, and it was inoculated on the LB plate for activation, and a single colony was picked to 20 mL of LB medium, and cultured at 37°C, 200 rpm overnight. Then, it was inoculated into a 5L fermenter with 2L of fermentation medium (initial medium) at a 1% inoculation rate, and cultured at 37°C, 300 rpm until OD 600 When the OD is about 0.8, add 0.1mM IPTG to induce the production of T7 RNA polymerase. Increase the rotation speed to control the dissolved oxygen to be greater than 30%. Since the culture medium contains buffer salt, there is no need to add additional ammonia water to regulate the pH. Continue to culture until the OD is 600 The fermentation was terminated when the pH value was 1.5-3. The cells were collected by centrifugation at 5000 g for 20 minutes at 4°C using a high-speed desktop centrifuge. The cells were then resuspended and washed three times with S30 buffer (10 mM Tris-AcOH, 14 mM magnesium acetate, 60 mM potassium acetate, 1 mM DTT, pH 8.2) to collect the cells.

[0043] Add 1 mL of S30 buffer to 1 g of bacteria, mix well, and use an ultrasonic disruptor to disrupt the bacteria at 800-900 bar for 3 times. Centrifuge the disrupted liquid at 4°C, 12000g for 30 minutes, and collect the supernatant. The collected supernatant is the cell extract, which is quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0044] (2) Cell-free protein expression reaction

[0045] The components of the cell-free protein expression system are shown in Table 1. The expression product is sfGFP green fluorescent protein, and the yield of sfGFP represents the activity of the cell extract.

[0046] Table 1 Cell-free protein expression system

[0047]

[0048]

[0049] The cell-free protein expression system was prepared according to Table 1, with a total volume of 50 μL, and the reaction vessel was a 2 mL round-bottom EP tube. The EP tube was placed in a constant temperature culture shaker at 30° C. and 220 rpm for 16 hours.

[0050] (3) Detection of cell-free protein expression yield

[0051] The sfGFP was recombinantly expressed and purified using conventional recombinant expression methods, and different concentrations (0-3 mg / mL) of sfGFP protein were prepared. The fluorescence value (Ex / Em 484 / 515) was measured using an ELISA reader, and a standard curve of protein concentration and fluorescence value was established, such as Figure 1 After the cell-free protein expression reaction was completed, the fluorescence value of the reaction solution was measured using an ELISA instrument, and the volume yield of the reaction product sfGFP protein was calculated based on the standard curve.

[0052] Example 2

[0053] Design of initial culture medium

[0054] (1) Selection of carbon source

[0055] Select glucose, glycerol, lactose and fructose, and prepare carbon source screening culture medium according to Table 2.

[0056] Table 2 Carbon source screening medium

[0057]

[0058]

[0059] The composition of the trace element and vitamin solution refers to the M9 inorganic salt medium formula, and three substances, biotin, microbial B12 and folic acid dihydrate, are added, as shown in Table 3.

[0060] Table 3 Trace element and vitamin solution formula

[0061]

[0062] The cell extract preparation method of Example 1 was used, wherein the fermentation medium was the carbon source screening medium shown in Table 2, and a batch fermentation parallel experiment was performed in four 5L fermentation tanks: temperature 37°C, initial rotation speed 300rpm, dissolved oxygen>30%, pH not controlled. 600When it reaches about 0.8, add IPTG at a final concentration of 0.1mM (IPTG is not added for cultures using lactose as the carbon source, and lactose can be used as an inducer to induce the expression of T7 RNA polymerase). Continue to culture until OD reaches about 2.0, sample 200mL of fermentation broth from the fermenter, use a high-speed desktop centrifuge at 4°C, 5000g for 20 minutes to collect the bacteria. Then resuspend and wash 3 times with S30 buffer to collect the bacteria. Add 1mL of S30 buffer to 1g of bacteria, mix well, and use an ultrasonic disruptor to disrupt the bacteria. Use a high-speed centrifuge at 4°C, 12000g for 30 minutes to centrifuge the broken liquid, and collect the supernatant as the cell extract. The prepared cell extract is used for cell-free protein expression reaction (the cell-free protein expression system is prepared using Example 1, as shown in Table 1), and the expression level of sfGFP is measured. Continue to culture until the stable growth period, OD 600 No longer growing.

[0063] The growth rates of E. coli under the four carbon source conditions were significantly different, such as Figure 2 As shown. With lactose and glycerol as carbon sources, the specific growth rate μ is significantly lower than that with glucose as carbon source, and the OD 600 The fermentation with fructose as the carbon source has a longer delay period than that with glucose as the carbon source, which makes the OD 600 Lower than glucose; in the logarithmic growth phase after the delay period, the specific growth rate μ is similar; but with fructose as the carbon source, the highest OD 600 , which is 49% higher than that of glucose as a carbon source. Glucose as a carbon source will produce a glucose effect, leading to the production of acetic acid, which in turn affects the growth of the bacteria and results in a final OD 600 Lower than the culture with fructose as carbon source.

[0064] The activity of the cell extract was characterized by measuring the sfGFP yield of the corresponding cell-free protein expression. The activity test results of the cell extracts prepared under four carbon source culture conditions are shown in Table 4. When glycerol and lactose were used as carbon sources, the specific growth rate μ was low, which directly affected the activity of the cell extract, and the sfGFP yields were 0.69 g / L and 0.83 g / L, respectively. Glucose was used as a carbon source and can be used as a positive control. The sfGFP yield was 1.55 mg / mL, which was in line with experimental expectations and at the same level as reported in the literature. With fructose as the carbon source, the specific growth rate μ in the logarithmic phase was basically the same as that when glucose was used as the carbon source, and its sfGFP yield reached 1.85 mg / mL, which was nearly 20% higher than the result with glucose as the carbon source. This result also proves that the activity of the cell extract is directly related to the specific growth rate μ. Based on the results of different carbon sources on bacterial growth and cell extract activity, fructose was selected as the carbon source for the next step of experimental optimization.

[0065] Table 4 Yield of sfGFP expressed in cell-free protein using different carbon sources

[0066]

[0067] (2) Optimization of carbon source concentration

[0068] Fructose was selected as the carbon source for E. coli culture, and then the concentration of fructose in the initial culture medium (based on the carbon source screening culture medium in Table 2) was investigated. Fructose culture media with concentrations of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, and 3.5% were prepared respectively, and other culture medium components were shown in Table 2. Parallel fermentation culture was carried out, and the cell extract was prepared by the method of step (1) of the present invention. In the logarithmic growth phase, i.e., OD 600 The results are shown in Table 5. When the fructose concentration is 2%, the logarithmic growth rate reaches a maximum value of 1.58h -1 At the same time, the yield of sfGFP reached 2.34 mg / mL. Therefore, 2% fructose concentration was selected for the next optimization experiment.

[0069] In the existing culture process, IPTG is also needed to induce the expression of T7 RNA polymerase. However, in order to simplify the operation, the present invention adds lactose to the initial culture medium as an auto-induction medium, and the addition amount is 0.1%-1%, preferably 0.5%. The addition of lactose has no significant effect on the biology of Escherichia coli, and there is no significant change in the activity of cell extracts.

[0070] Table 5 Fermentation results of different fructose concentrations

[0071]

[0072]

[0073] In summary, the optimal initial culture medium formula of the present invention is shown in Table 6.

[0074] Table 6 Initial culture medium formula

[0075]

[0076] Example 3

[0077] Batch-fed-batch fermentation - optimization of feeding strategy

[0078] (1) Feed medium composition

[0079] Batch-fed fermentation is a general method for obtaining high-density bacteria, that is, after the initial culture medium carbon source is consumed, the carbon source and nitrogen source are added to maintain the high-speed growth of the bacteria and reach a higher cell density. In addition, in order to obtain a cell extract with the same high activity as low-density fermentation, the bacteria must be maintained in the logarithmic growth phase and a larger specific growth rate. Therefore, it is considered that the carbon source feed is fructose + magnesium sulfate, and the nitrogen source feed is a nitrogen source solution, as shown in Table 7 and Table 8, respectively.

[0080] In the initial medium formula (shown in Table 6), in order to support the rapid growth of E. coli, a nitrogen source solution of yeast powder and yeast peptone was added. Its composition is shown in Table 7.

[0081] Table 7 Nitrogen source solution formula

[0082]

[0083] The novel culture medium used in the present invention is improved based on M9 inorganic salt culture medium. First, the type and concentration of carbon source are screened, and a mixed carbon source of fructose and lactose is selected, wherein fructose is used as the main carbon source and lactose is used as an inducer. The addition amount of inorganic salts, trace elements and vitamins is adjusted. Nitrogen source solution (i.e. yeast powder and yeast peptone) is added to the initial culture medium to support the rapid growth of Escherichia coli.

[0084] The optimized feeding strategy of the present invention is as follows: after the carbon source (fructose and lactose) in the initial culture medium (i.e., the optimal fermentation medium formula described in Table 6) is consumed, the feeding is started immediately. The feeding ingredients are the carbon source, i.e., fructose and magnesium sulfate solution, and the nitrogen source, i.e., the nitrogen source solution shown in Table 7. The formula of the feeding carbon source culture medium is shown in Table 8,

[0085] Table 8 Carbon source feed formula

[0086]

[0087] (2) Optimization of feeding rate

[0088] Batch-fed-batch fermentation was performed. When the initial carbon source was exhausted, the dissolved oxygen increased rapidly. At this time, 10 ml of carbon source was added at one time, and the time of feeding was recorded. When the added carbon source was exhausted, the dissolved oxygen increased rapidly again, and the time at this time was recorded. The consumption rate of the carbon source was calculated to be approximately 0.33 ml / L / min. Therefore, the initial carbon source feed rate was set to 0.33 ml / L / min, and the initial nitrogen source feed rate was set to 0.1 mL / L / min, which was always maintained at 30% of the carbon source.

[0089] After the feeding starts, in order to maintain the exponential growth of Escherichia coli, the feeding rate of carbon source and nitrogen source increases at an exponential rate, and the exponential growth rate is k (k = ln (N2 / N1) / (t2-t1), N1 and N2 are the feeding rates at t1 and t2, t1 and t2 represent any two times, and the unit is hour). Among them, the exponential growth rate k is the trend of the addition rate. The addition rate will change with the change of time. The addition rate corresponding to each time is different. From the initial feeding rate, it increases at the exponential rate k. The dissolved oxygen is controlled to be greater than 30% by adjusting the rotation speed and ventilation; the pH is controlled to 6.5±0.1 by ammonia water. Record OD during the fermentation process 600 The natural logarithm of the change and the culture time are plotted. Figure 3 When OD 600 After reaching 60, the fermentation was stopped, and the cell extract was prepared. The activity of the cell extract was measured using the cell-free protein expression system of Example 1. The experimental data are shown in Table 9.

[0090] Table 9 Fermentation results at different feeding rates

[0091]

[0092] As shown in Table 9, the exponential growth of E. coli can be maintained by the exponential growth of the feeding rate; the exponential rate of feeding is positively correlated with the exponential growth rate of E. coli. The larger the exponential rate of feeding, the larger the specific growth rate of the bacteria; the specific growth rate μ of the bacteria is slightly lower than the exponential rate of feeding k. The yield of sfGFP reflects the activity of the cell extract and is positively correlated with the specific growth rate of the bacteria; when the exponential feeding rate k is 0.9h -1 , the yield of sfGFP reached 2.53 mg / mL, which was slightly higher than the activity of the cell extract obtained by low-density fermentation of fructose.

[0093] Based on the above data, the exponential rate of feeding is selected as 1.0h -1 Under this parameter, the cell density of Escherichia coli OD 600 It can reach 60 and maintain high activity of cell extraction, and the yield of sfGFP can reach more than 2 mg / mL.

[0094] Example 4

[0095] Comparison with the commonly used 2YTPG medium

[0096] (1) Batch-fed-batch high-density fermentation of 2TYPG medium

[0097] In the current public reports (Levine et al. J Vis Exp 25:(144), Kwon et al. Sci Rep 5:8663), the culture medium commonly used for preparing E. coli cell extracts required for cell-free protein synthesis is 2YTPG, and its composition is shown in Table 10. When using 2YTPG culture medium to prepare cell extracts, E. coli is generally cultured in a shake flask until OD 600 After reaching 1-3 growth phases, the bacteria are harvested to prepare cell extracts. No reports have been seen on batch-fed-batch fermentation using 2YTPG medium. To demonstrate the advanced nature of the medium formulation and feeding strategy of the present invention, it was compared with batch-fed-batch fermentation using 2YTPG medium.

[0098] According to the formula in Table 10, prepare the initial culture medium of a 5L tank. Fermentation began after inoculation, the temperature was 37°C, the dissolved oxygen was controlled to be greater than 30% by the stirring speed and ventilation volume, and the pH was controlled at 6.5±0.1 by adding ammonia water. When the dissolved oxygen rose rapidly, it proved that the initial carbon source was consumed, and carbon source (500g / L glucose + 13.3g / L magnesium sulfate) and nitrogen source solution (50g / L yeast powder and 100g / L yeast peptone) were added. The initial carbon source feed rate was 0.33ml / L / min, and the initial nitrogen source feed rate was 0.1mL / L / min, and then the exponential rate k=1.0h -1 improve.

[0099] Table 10 2YTPG medium formula

[0100]

[0101] (2) Fermentation results of 2YTPG medium

[0102] Sampling during fermentation to measure OD 600 The natural logarithm of the culture time was plotted. Figure 4 In the fermentation using 2YTPG, the specific growth rate μ continued to decrease during the fed-batch culture phase and could not maintain growth in the logarithmic growth phase; after 9 hours of culture, OD 600 Using the culture medium and feeding strategy of this patent, the growth of E. coli can be maintained in the logarithmic growth phase, and the OD 600 2YTPG medium uses glucose as a carbon source. During batch culture and fed-batch culture, glucose concentration may accumulate, resulting in a glucose effect on the growth rate and the final OD 600 Produced a significant inhibitory effect.

[0103] The activities of the cell extracts prepared by the two fermentation methods were compared, and sfGFP was expressed using a cell-free protein expression system. The results are shown in Table 11. The cell extract prepared by 2YTPG medium expressed sfGFP at an output of only 0.52 mg / mL, which was significantly different from the expression of 2.35 mg / mL of the cell extract prepared by the present invention. Figure 5 As shown, from left to right are: a negative control of a cell-free system without a DNA template, a 2YTPG culture medium, and the present invention. It can be seen that the cell extract prepared by the present invention expresses a higher concentration of sfGFP after the reaction, proving that the culture medium of the present invention has a better effect than the commonly used 2YTPG culture medium.

[0104] Table 11 Comparison of the activity of 2YTPG culture medium and cell extracts of this patent

[0105]

[0106] Example 5

[0107] Scale-up experiment of 100L fermenter

[0108] The above batch-fed-batch fermentation process was scaled up to a 100-L fermenter. The activated single colony was picked into 20 mL of LB medium and cultured overnight at 37°C, 200 rpm. A 5-L shake flask containing 2 L of LB medium was inoculated at 1% of the inoculum and cultured at 37°C, 250 rpm until OD 600 The 2L seed solution was transferred to a 100L fermentation tank, which contained 60L of the optimal fermentation medium of the present invention, the formula of which is shown in Table 6.

[0109] The fermentation temperature was 37°C, the initial stirring rate was 150 rpm, the dissolved oxygen was controlled to be greater than 30% by increasing stirring and ventilation, and the pH was controlled at 6.5±0.1 by adding ammonia water. When the dissolved oxygen rose rapidly, it proved that the initial carbon source was consumed, and carbon and nitrogen sources were added. The initial carbon source addition rate was set to 19.8 ml / min, and the exponential rate k=1.0 h -1 The nitrogen source feed rate was maintained at 30% of the carbon source. Samples were taken to measure the OD during the fermentation process. 600 And record, when OD 600 When the temperature reaches 60, stop fermentation and cool down to below 15°C. Prepare cell extracts by adding 1 mL of S30 buffer to 1 g of bacteria, mix well, and use a high-pressure homogenizer to break the bacteria at 800-900 bar for 3 times. The broken liquid is centrifuged in a high-speed centrifuge at 4°C and 12000g for 30 minutes. The supernatant is collected as the cell extract and cell-free protein expression of sfGFP is performed. The fermentation curve of the 100L fermenter is shown in Figure 2. Figure 6As shown in the figure, the changes in parameters such as dissolved oxygen, pH, and stirring speed were in line with expectations, and the process was successfully scaled up from 5L to 100L. 600 Changes over time such as Figure 7 As shown, the specific growth rate μ reached 1.09, and the final OD 600 The yield of sfGFP expressed in cell-free protein was 2.38 mg / mL, which achieved good process scale-up.

[0110] Example 6

[0111] Cell extracts prepared by high-density fermentation are used to express the toxic protein DNase I in a cell-free protein expression system.

[0112] (1) Construction of DNase I expression plasmid

[0113] The cell-free protein expression system is suitable for expressing toxic proteins because there are no living cells in the expression system, which avoids the toxic effects of toxic protein expression on cells. In the previous example, the reporter protein sfGFP was expressed using the cell-free protein expression system. In this example, a toxic protein, DNase I, was selected for expression to verify the applicability of the cell-free protein expression system. The DNA sequence of DNase I after codon optimization was constructed into the pJL1 vector (such as SEQ ID NO.2). Figure 8 The vector construction of DNase I is shown. The pJL1-DNase I plasmid contains a T7 promoter and RBS, and an OmpA signal peptide is present at the N-terminus of DNase I to increase the expression of the target gene.

[0114] (2) Cell-free expression and activity determination of DNase I

[0115] The cell extract prepared by high-density fermentation in a 100L tank was treated with 0.1mM iodoacetamide for 30 minutes; 14μM disulfide isomerase DsbC, 4mM oxidized glutathione, and 1mM reduced glutathione (all final concentrations) were added to the cell-free protein expression system. Using the constructed pJL1-DNase I plasmid as a template, cell-free protein expression was performed according to the cell-free protein expression system of Example 1. SDS-PAGE electrophoresis was performed after the reaction, and the results are shown in Fig. 9 As shown, there is obvious protein expression, namely DNase I, around 30kD.

[0116] Crude enzyme activity determination of cell-free protein expressed DNase I: In a 20 μL reaction system, 200 ng of substrate λDNA, 1 μL of cell-free reaction solution with different dilutions were added, and the reaction was carried out at 37°C for 10 min. The negative control was the original cell-free reaction solution without template DNA. After the reaction, nucleic acid electrophoresis was performed, and the results were shown in Fig.10 It can be seen that after diluting the cell-free reaction solution 1000 times, the substrate λDNA can still be completely digested. The DNase I expressed by the cell-free protein has a very high activity.

Claims

1. An Escherichia coli culture medium, characterized in that The culture medium includes an initial culture medium or an initial culture medium and a feed culture medium, wherein the initial culture medium includes fructose, lactose, magnesium sulfate, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, citric acid, trace elements and vitamin solution, and a nitrogen source, and the feed culture medium includes a carbon source and a nitrogen source, wherein the carbon source is fructose and a magnesium sulfate solution, and the nitrogen sources in the initial culture medium and the feed culture medium are both nitrogen source solutions containing added yeast powder and yeast peptone.

2. The Escherichia coli culture medium according to claim 1, characterized in that The initial culture medium comprises 1-3% by mass of fructose, 0.1-1% of lactose, 0.1-0.5% of magnesium sulfate, 0.1-0.4% of ammonium sulfate, 0.5-5% of dipotassium hydrogen phosphate, 0.2-3% of potassium dihydrogen phosphate, 0.1-2% of citric acid, 0.01%-0.5% of trace element and vitamin solution, and 0.5-2% of nitrogen source solution.

3. The Escherichia coli culture medium according to claim 1, characterized in that The trace element and vitamin solution comprises copper sulfate pentahydrate, zinc chloride, ferrous sulfate heptahydrate, anhydrous cobalt chloride, calcium chloride dihydrate, manganese sulfate monohydrate, biotin, vitamin B12, and folic acid dihydrate.

4. The Escherichia coli culture medium according to claim 3, characterized in that The concentrations of the trace elements and vitamin solutions are: copper sulfate pentahydrate 0.1-2g / L, zinc chloride 0.1-0.7g / L, ferrous sulfate heptahydrate 1-5g / L, anhydrous cobalt chloride 0.2-4g / L, calcium chloride dihydrate 0.5-6g / L, manganese sulfate monohydrate 0.5-5g / L, biotin 0.05-0.2g / L, vitamin B12 0.005-0.02g / L, and folic acid dihydrate 0.01-0.2g / L.

5. The Escherichia coli culture medium according to claim 1, characterized in that The fructose concentration in the carbon source is 300-600 g / L, and the magnesium sulfate concentration is 10-20 g / L.

6. The Escherichia coli culture medium according to claim 1, characterized in that The concentration of yeast powder in the nitrogen source solution is 20-100 g / L, and the concentration of yeast peptone is 50-150 g / L.

7. Use of the Escherichia coli culture medium according to claim 1 in preparing cell extracts.

8. Use of the Escherichia coli culture medium according to claim 1 in preparing a cell extract required for cell-free protein synthesis.

9. The use according to claim 7 or 8, characterized in that: The application process includes the following steps: (1) Batch-fed-batch fermentation was carried out in a fermenter containing Escherichia coli at elevated temperature. When the carbon source in the initial culture medium was exhausted and the dissolved oxygen increased rapidly, carbon source and nitrogen source were added and the OD was measured. 600 , stop fermentation, centrifuge, resuspend and wash to collect the cells; (2) Add the bacterial cells prepared in step (1) into S30 buffer solution, crush them, and centrifuge them to collect the supernatant, which is the cell extract.

10. The use according to claim 9, characterized in that: In step (1), the amount of carbon source and nitrogen source added is 10-15 ml respectively, the carbon source addition rate is 0.2-0.45 ml / L / min, and the nitrogen source addition rate is 20-40% of the carbon source.

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