Quantitative method for E. coli cell sonication disruption based on firefly luciferase

Abstract

This invention relates to a quantitative method for ultrasonic disruption of *E. coli* cells, specifically a method based on firefly luciferase in *E. coli* cells. The method involves mixing *E. coli* expressing firefly luciferase as an internal standard with a suspension of target protein-expressing bacteria, followed by ultrasonic disruption of the cells. The degree of disruption of the target protein-expressing bacterial suspension is quantitatively calculated by measuring the activity of the firefly luciferase. The target protein-expressing bacterial suspension uses *E. coli* expressing the target protein. Compared with existing technologies, this invention overcomes the shortcomings of complex experiments, low efficiency, and unstable accuracy in existing technologies. It achieves efficient and highly accurate quantification of the degree of ultrasonic disruption of *E. coli* cells, reduces experimental complexity, and improves research and development efficiency and accuracy. This provides a scientific basis and new approach for the development of new feed protein resources and livestock breeding research, and has broad application potential.

Description

Technical Field

[0001] This invention relates to a quantitative method for ultrasonic disruption of Escherichia coli cells, specifically a quantitative method for ultrasonic disruption of Escherichia coli cells based on firefly luciferase. Background Technology

[0002] In E. coli-based exogenous protein expression systems, the target proteins for isolation and purification often exist in the form of intracellular components. During the isolation and extraction process, cell disruption or lysis is essential to release intracellular contents. Therefore, cell disruption becomes a key technology for extracting intracellular bioactive substances.

[0003] Currently, commonly used techniques for disrupting E. coli cells include high-pressure homogenization, high-speed bead milling, ultrasonic disruption, enzymatic dissolution, and chemical permeation. Among these, ultrasonic disruption is widely used as a low-cost and easy-to-use method. This method utilizes the cavitation effect induced by ultrasound in a liquid medium to generate high-pressure shock waves and localized high temperatures, thereby disrupting the cell wall's network structure and releasing the cell contents, which can then participate in subsequent purification processes.

[0004] The effectiveness of ultrasonic disruption is influenced by a synergistic effect of factors such as probe diameter, ultrasonic power, solution volume, container size, container material, disruption buffer composition, and ultrasonic treatment time. It often requires repeated optimization of specific disruption conditions to achieve the appropriate disruption effect—ensuring a high disruption rate while preserving as much of the target protein's biological activity as possible. Therefore, optimizing ultrasonic disruption parameters has become a crucial step in the purification of prokaryotically expressed proteins. However, during optimization, determining the degree of cell disruption often relies on SDS-PAGE and subsequent staining (a complete electrophoresis and staining process takes at least 16 hours), which is time-consuming, labor-intensive, and only yields semi-quantitative results, highlighting the need for improved efficiency and accuracy. More importantly, to ensure the activity of the obtained product (a large protein product quantity does not necessarily equate to high activity), researchers often need to verify the product activity using various methods, a process that also requires significant manpower and time. For example, the verification methods used for commonly used enzymes in biological research are as follows:

[0005] (1) Restriction endonuclease: Its activity refers to the ability of one unit of restriction endonuclease to completely digest 1 μL of a specific substrate in a 50 μL system within 1 hour under optimal reaction conditions. This means that when the target product is a restriction endonuclease, its activity detection (including substrate treatment and subsequent detection processes) usually takes more than 3 hours.

[0006] (2) Nitrogenase: The commonly used method for detecting its activity is the "acetylene reduction method," which utilizes nitrogenase to reduce acetylene gas to ethylene gas. Although the principle of detection is relatively simple, in actual operation, it requires not only about 60 minutes of incubation at a constant temperature (30°C) in the dark, but also the detection of product formation by gas chromatography. The gradual cooling of the gas chromatography column from startup to stabilization and then to the end of detection often takes 3 to 5 hours. If the gas chromatography column needs to be cleaned and aged during this period, an additional 4 to 12 hours are required.

[0007] (3) RNA polymerase: Its activity can be detected using isotope labeling or fluorescence methods. Using isotope labeling to determine enzyme activity can easily lead to radioactive contamination, requires sophisticated equipment, and incurs high operating and post-operative costs. Using fluorescence methods, the products synthesized by RNA polymerase are easily degraded by RNase, affecting the detection results, and places high demands on the testing personnel and environment. Therefore, both methods have a certain learning curve.

[0008] As can be seen from the above, the existing methods for detecting the protein activity of Escherichia coli after ultrasonic disruption are all specific to certain groups, and the detection procedures are not standardized and are mostly quite complex or difficult to process. This not only results in low efficiency but also easily affects the accuracy of the detection results. Therefore, it is necessary to propose an appropriate method to simplify the detection method and improve the accuracy of the data. Summary of the Invention

[0009] The purpose of this invention is to provide a quantitative method for ultrasonic disruption of Escherichia coli cells based on firefly luciferase to solve at least one of the above-mentioned problems. This method overcomes the shortcomings of existing technologies, such as complex detection experiments, low efficiency, and unstable accuracy. It achieves efficient and accurate quantification of the degree of ultrasonic disruption of E. coli cells, thereby reducing experimental complexity, improving research and development efficiency and accuracy, and providing a scientific basis and new approach for the development of new feed protein resources and livestock and poultry breeding research and development. It has broad application potential.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A quantitative method for ultrasonic disruption of Escherichia coli cells based on firefly luciferase involves mixing Escherichia coli expressing firefly luciferase as an internal standard with a suspension of target protein-expressing bacteria, followed by ultrasonic disruption of the cells. The degree of disruption of the target protein-expressing bacterial suspension is quantitatively calculated by measuring the activity of firefly luciferase.

[0012] The target protein is expressed in the bacterial suspension containing Escherichia coli.

[0013] Preferably, the quantitative method includes the following steps:

[0014] S1. Induction of target protein expression: Escherichia coli was added to the first culture medium and cultured with shaking once. The culture product was then transferred to the second culture medium and cultured with shaking a second time. IPTG was added to the second culture product and cultured for a second time to obtain the bacterial culture after induction of expression.

[0015] The first culture medium and the second culture medium are ampicillin-resistant LB medium;

[0016] S2. Ultrasonic disruption of bacterial cells: Centrifuge the bacterial suspension obtained in step S1 after induction and collect the bacterial sludge, then resuspend it in PBS buffer. Repeat centrifugation and collection of bacterial sludge, then resuspend it in PBS buffer. Mix the internal standard into the bacterial suspension, then perform ultrasonic disruption and centrifugation to obtain the supernatant.

[0017] S3. Detection of firefly luciferase activity: Take the supernatant obtained in step S2 for protein activity detection.

[0018] Preferably, the Escherichia coli is a glycerol bacterium, and the volume ratio of Escherichia coli to the first culture medium is 1-5:750; the first shaking culture is carried out at 37°C and 220 r / min for 16 h.

[0019] Preferably, the volume ratio of the primary culture product to the secondary culture medium is 500 μL: 150 mL; the secondary shaking culture is carried out at 37°C and 220 r / min until the turbidity of OD650 is 0.5 to 0.6.

[0020] Preferably, the final concentration of IPTG is 50–100 μM; the continued culture is carried out at 15°C and 220 r / min for 20–24 h.

[0021] Preferably, the centrifugation is performed at 6000-8000g and 0-4℃ for 2-5 minutes; the resuspension is performed using PBS buffer pre-cooled at 0-4℃, wherein the pH of the PBS buffer is 7.4 and the concentration is 0.01mol / L.

[0022] Preferably, the volume ratio of the internal standard to the target protein expression bacterial suspension is 1:100 to 1000.

[0023] Preferably, the ultrasonic disruption is carried out in an ice-water mixture incubation process, using pulse disruption. The ultrasonic disruption power is 160–240 W, and the pulse conditions are: disruption for 2 seconds, pause for 3 seconds, and disruption time for 10–20 minutes.

[0024] Preferably, the post-centrifugation is performed at 15000-18000g and 0-4℃ for 10-15 minutes.

[0025] Preferably, the luciferase detection is performed using an enzyme-linked immunosorbent assay (ELISA) reader with chemiluminescence detection capability.

[0026] The working principle of this invention is as follows:

[0027] Firefly luciferase (FLuc) is abundant in the tail of fireflies and is closely related to bioluminescence. With a molecular weight of approximately 61 kDa, it is commonly used for reporter gene detection due to its high sensitivity, short half-life, and strong specificity, making it an important marker tool in medical and life science research. The substrate D-Luciferin, which reacts with FLuc, carries a negative charge under alkaline conditions and cannot penetrate the cell wall and membrane of *E. coli*, thus failing to react with intracellular FLuc. Therefore, if the pH of the buffer solution is controlled within the alkaline range, when the substrate is added to a bacterial suspension expressing FLuc, the substrate only reacts with the free FLuc in the buffer solution, emitting fluorescence. This can be used to characterize the damage to the outer barrier during bacterial lysis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This method utilizes *E. coli* expressing FLuc as an internal standard, mixed with the bacterial suspension to be lysed before disruption. The degree of cell disruption is characterized by the release of luciferase activity during disruption (changes in luciferase activity in the supernatant after disruption). Due to the extremely high detection sensitivity and ease of use of firefly luciferase, this method requires only a small amount of bacterial cells for quantification while maintaining high accuracy. It is particularly suitable for situations where target protein activity detection is difficult, providing quantitative guidance for optimizing ultrasonic disruption conditions of *E. coli*, significantly improving research efficiency, reducing experimental complexity, and enhancing both efficiency and accuracy. This offers a scientific basis and new approach for developing new feed protein resources and for livestock and poultry breeding research, demonstrating broad application potential.

[0030] This method typically takes no more than 15 minutes to detect and can be fully automated using an autosampler. It is extremely easy to operate and has excellent detection efficiency. Therefore, using the release of luciferase activity to calibrate the concentration and activity of other proteins in the lysis buffer can reduce the complexity of experiments and improve research and development efficiency.

[0031] This method uses a firefly luciferase vector based on the E. coli cspA promoter, making most E. coli strains usable as hosts. Furthermore, the vector does not contain common isolation and purification tags, and the internal standard has no effect on the purification of the final product, thus possessing the potential for widespread use.

[0032] In actual ultrasonic disruption processes, the further away from the ultrasonic probe, the lower the thermal effect. Therefore, even when using disruption devices equipped with temperature probes, it is difficult to effectively evaluate the overall temperature of the solution. FLuc, with its poor thermal stability (a significant decrease in activity can be detected above 35°C) and moderate molecular weight, is well-suited for monitoring the severity of disruption conditions and can provide an overall quantitative mean of the solution, offering a reference for preserving protein activity during disruption. This data is particularly valuable for target proteins with higher stability than FLuc, facilitating the maximization of target protein recovery efficiency.

[0033] The bacterial cells containing firefly luciferase can be stored at -80℃ for at least 90 days without a significant decrease in enzyme activity, and the cells' tolerance to ultrasonic disruption is not altered by cryopreservation. They can be directly thawed and mixed with target cells before use to assist in quantitative analysis. They are convenient to use and have good application prospects.

[0034] Therefore, this quantitative method is a simple, stable and efficient way to assist in the quantitative determination of the degree of ultrasonic disruption of Escherichia coli. It provides a scientific basis and new approach for the development of new feed protein resources and livestock and poultry breeding research, and further promotes the technological innovation and application development of the feed industry and the breeding industry. Attached Figure Description

[0035] Figure 1 The diagram shows the structures of the pUCC-FLuc, pUCC-RLuc, pUCC-EGFP, and pUCC-mCherry expression vectors in the examples.

[0036] Figure 2 The standard curves for the activities of luciferase and fluorescent protein in the examples are shown (n=3).

[0037] Figure 3 The figure shows the results of the quantitative test of firefly luciferase-assisted quantification in the examples (n=3);

[0038] Figure 4 The figure shows the results of the sensitivity test of firefly luciferase to ultrasonic disruption power in the example (n=3);

[0039] Figure 5 This is a graph showing the effect of firefly luciferase on target protein purification in the examples;

[0040] Figure 6 The figure shows the effect of low-temperature freezing on firefly luciferase in the examples (n=3). Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0042] 1. The strains and main reagent sources used in the following examples:

[0043] BL21 chemocompetent cells: Shanghai Weidi Biotechnology Co., Ltd.; After transformation into BL21 competent cells, the expression vectors pUCC-FLuc, pUCC-RLuc, pUCC-EGFP, and pUCC-mCherry, used to express firefly luciferase (FLuc), Renilla luciferase (RLuc), green fluorescent protein (EGFP), and red fluorescent protein (mCherry), are shown in the following structures: Figure 1 As shown in a and b in the figure.

[0044] Ampicillin sodium salt (used to prepare ampicillin-resistant medium), RealBand trichrome prestained protein marker (used for calculating protein molecular weight by electrophoresis), IPTG: Sangon Biotech (Shanghai) Co., Ltd.

[0045] 10×SDS electrophoresis buffer (for protein detection by electrophoresis), 10% protein precast gel (for separating proteins of different molecular weights by electrophoresis), Ni-NTA resin (nickel beads): Beijing Lanbolide Trading Co., Ltd.

[0046] DL101-01 Dual Luciferase Reporter Gene Detection Kit: Nanjing Novizan Biotechnology Co., Ltd.

[0047] All other reagents used in this method, unless otherwise specified, can be commercially available reagents commonly used in the field.

[0048] 2. The experimental method in the following example:

[0049] 2.1 Activation of bacterial strains and induction of target protein expression

[0050] Add 1–5 μL of frozen glycerol bacteria to 750 μL of ampicillin-resistant LB medium (10 g tryptone, 5 g yeast extract, 10 g sodium chloride dissolved in distilled water and brought to a final volume of 1 L, pH adjusted to 7.2–7.4, the same below), using 1.5 mL centrifuge tubes as containers, and incubate at 37°C and 220 rpm with shaking for 16 hours. Then, take 500 μL of the culture and add it to 150 mL of ampicillin-resistant LB medium, and continue incubating at 37°C and 220 rpm with shaking until the turbidity (OD650) is between 0.5 and 0.6. Then, add IPTG to a final concentration of 50–100 μM, and incubate at 15°C and 220 rpm for 20–24 hours before proceeding with subsequent detection.

[0051] 2.2 Ultrasonic disruption of bacterial cells

[0052] After inducing expression as described in 2.1, the bacterial culture is centrifuged at 6000-8000g and 0-4℃ for 2-5 minutes. The supernatant is discarded, and the bacterial sludge is collected. The sludge is resuspended in 15-25 mL of pre-cooled (0-4℃, the same below) PBS buffer (pH 7.4, 0.01 mol / L, the same below). The centrifugation is repeated once more. After discarding the supernatant, the bacterial cells are resuspended in 10-20 mL of pre-cooled PBS.

[0053] The bacterial suspension was then placed in a 50 mL centrifuge tube in an ice-water mixture. Using a 6 mm diameter ultrasonic probe, the cell disruption conditions were set to "disrupt for 2 seconds, stop for 3 seconds" and the suspension was disrupted using a cell disruptor at the specified power. The disrupted bacterial suspension was then centrifuged at 15000–18000 g at 0–4 °C for 10–15 min, and the supernatant was collected to detect protein activity.

[0054] 2.3 Luciferase activity detection

[0055] Luciferase detection was performed using a commercially available dual-luciferase (FLuc / RLuc) assay kit, strictly following the instructions. Luciferase samples were pre-diluted with PBS (enough to prevent detector overexposure due to the catalytic reaction). Then, 20–30 μL of the diluted sample was added with the cell lysis buffer provided with the kit, and the sample was automatically added to each well using a multi-mode microplate reader. During detection, an appropriate data attenuation value should be set according to the substrate concentration and the performance of the instrument to avoid overexposure.

[0056] 2.4 Detection of fluorescent protein activity

[0057] When detecting the activity of EGFP (filter combination: EX-485; EM-535) and mCherry (filter combination: EX-535; EM-595) using a multi-functional microplate reader, take the supernatant after disruption, add PBS to make up to 100-150 μL, and then perform the detection. To avoid overexposure, an appropriate attenuation value should be set during detection.

[0058] 2.5 Detection of the quantitative effect of firefly luciferase-assisted fragmentation

[0059] Following the steps described in 2.1, bacterial suspensions expressing four target proteins (FLuc, RLuc, EGFP, and mCherry) were induced. Subsequently, each bacterial suspension was centrifuged and washed as described in 2.2 to obtain 10–20 mL of concentrated bacterial suspensions expressing each of the four target proteins. Then, concentrated bacterial suspension expressing FLuc (internal standard) was added to the bacterial suspensions containing RLuc, EGFP, and mCherry (target protein expression suspensions) at a volume ratio of 1 / 100–1 / 1000 (depending on FLuc expression levels, substrate activity, and instrument sensitivity) as an auxiliary quantitative internal standard for cell disruption. Cell disruption was then performed at 160 W. After disruption, the supernatant was used to detect the corresponding luciferase and fluorescent protein activities.

[0060] The extracted protein was characterized using RLuc, EGFP, and mCherry, which are equally convenient for activity detection, and its activity release curve was observed to determine if it was consistent with that of FLuc. If they were consistent, it indicated that the lysis process of the FLuc-expressing bacteria was the same as that of the target protein-expressing bacteria, and the quantification method was effective.

[0061] 2.6 Evaluation of the effect of different crushing powers on luciferase activity

[0062] Following the steps described in 2.1, the bacterial suspension expressing the target protein was induced and centrifuged at 6000–8000 g at 0–4 °C for 2–5 min to wash the bacterial cells. The cells were then resuspended in 15–25 mL of pre-chilled PBS and centrifuged again at 6000–8000 g at 0–4 °C for 2–5 min. The supernatant was discarded, and the cells were resuspended in 10–20 mL of pre-chilled PBS. 10–50 μL each of the resuspended bacterial suspensions expressing FLuc and RLuc were added to 10–20 mL of pre-chilled PBS, mixed thoroughly, and then the cells were disrupted using the ultrasonic disruption method described in 2.2 with different ultrasonic powers. The disrupted bacterial suspensions were centrifuged at 15000–18000 g at 0–4 °C for 10–15 min, and the luciferase activity in the supernatant was measured.

[0063] 2.7 Protein purification and electrophoretic detection using the nickel bead method

[0064] Wash the nickel beads three times with 10-20 times their volume of PBS (select washing conditions according to the instructions). Add an equal volume of nickel beads to the protein solution (sample to be purified), mix well, and store the sample. Then, vortex at 15-20 rpm and 4-8°C for 10-12 hours, centrifuge at 400-600g for 3-5 minutes, and store 50-100 μL of the supernatant. Wash the nickel beads three more times with PBS, and store 20-50 μL of the nickel beads. Add 1× SDS-PAGE buffer to each sample, bringing the volume to 100-150 μL. After sample preparation, take 10-20 μL for SDS-PAGE (10% separating gel concentration) and perform protein staining analysis using the Cochlear staining method.

[0065] 2.8 Disruption and Detection of Frozen Bacteria

[0066] Following the steps described in 2.1, induce bacterial expression of the target protein, centrifuge and wash the cells once, then resuspend in 10–20 mL of PBS containing 20–30% (v / v) glycerol. Aliquot the resuspended bacterial suspension and freeze at -75–-80°C. Subsequently, every 30 days, take 1.0–2.0 mL each of the bacterial suspension containing FLuc and RLuc, thaw in a water bath at 30–35°C, centrifuge, and resuspend in 10–20 mL of pre-chilled PBS, followed by lysis at 160W. After centrifuging at 15000–18000 g at 0–4°C for 10–15 min, collect the supernatant to detect luciferase activity.

[0067] Example 1

[0068] 1 μL of the frozen bacterial suspension was added to 750 μL of ampicillin-resistant LB medium. The medium was then cultured in 1.5 mL centrifuge tubes at 37°C and 220 rpm with shaking for 16 hours. Subsequently, 500 μL of this suspension was added to 150 mL of ampicillin-resistant LB medium and cultured at 37°C and 220 rpm with shaking until the turbidity (OD650) was between 0.5 and 0.6. IPTG was then added to a final concentration of 100 μM, and the medium was cultured at 15°C and 220 rpm for another 24 hours before subsequent detection.

[0069] Following the steps above, we obtained bacterial cultures that induced FLuc expression, RLuc expression, EGFP expression, and mCherry expression in sequence.

[0070] In this embodiment, the specific experimental parameters for step 2.2 are as follows: After centrifuging the induced bacterial suspension at 7000g and 4℃ for 2 min, the supernatant is discarded, the bacterial sludge is collected, and the suspension is resuspended in 20mL of pre-chilled PBS buffer. This process is repeated once more, and the supernatant is discarded. The bacterial cells are then resuspended in 10mL of pre-chilled PBS. Subsequently, the bacterial suspension is placed in a 50mL centrifuge tube in an ice-water mixture. Using a 6mm diameter ultrasonic probe, the pulse disruption conditions are set to "disrupt for 2 seconds, stop for 3 seconds," and the cells are disrupted using a cell disruptor at the specified power. After disruption, the bacterial suspension is centrifuged at 15000g and 4℃ for 15 min, and the supernatant is used to detect protein activity.

[0071] 1. Determination of detection conditions for luciferase and fluorescent protein activities:

[0072] This experiment first employed a single-factor design to examine the target protein expression of four different bacterial strains obtained in the examples. Following the steps described in 2.3 and 2.4, the target protein activity in the centrifuged supernatant after ultrasonic disruption (160W, 20min, 2s / 3s intermittent disruption) was measured, and standard curves were plotted (Table 1 and 2). Figure 2 , Figure 2 In the table, a, b, c, and d correspond to FLuc, RLuc, EGFP, and mCherry, respectively.

[0073] As can be seen from the standard curve, the amount of supernatant added in this experiment (the supernatant was diluted 500 times with PBS before the FLuc and RLuc activity detection) has a strong linear relationship with the reaction fluorescence intensity, which can be considered that the detection method and the amount of supernatant used are effective and accurate.

[0074] Table 1. Standard curve data for protein activity (n=3)

[0075]

[0076] 2. Test on the effectiveness of ultrasound-mediated luciferase-assisted quantification of firefly fragments:

[0077] In this embodiment, the specific experimental parameters for step 2.5 are as follows: After inducing bacterial expression, the bacterial suspension is centrifuged at 7000g and 4℃ for 2 minutes. The supernatant is discarded, and the bacterial sludge is collected. The sludge is resuspended in 20mL of pre-chilled PBS buffer, and the centrifugation is repeated once more. After discarding the supernatant, the bacterial cells are resuspended in 10mL of pre-chilled PBS. 10mL of concentrated bacterial suspensions expressing each of the four target proteins are obtained. Subsequently, concentrated bacterial suspension expressing FLuc is added to the bacterial suspensions containing RLuc, EGFP, and mCherry at a volume ratio of 1 / 500 as an auxiliary quantitative internal standard for disruption. During disruption, the bacterial suspension is placed in a 50mL centrifuge tube in an ice-water mixture. A 6mm diameter ultrasonic probe is used, and the pulse disruption conditions are set to "disrupt for 2 seconds, stop for 3 seconds" at 160W power using a cell disruptor. After disruption, the bacterial suspension is centrifuged at 15000g and 4℃ for 15 minutes, and the supernatant is used to detect protein activity.

[0078] Using the average value of the most active protein in the fragmented sample as 100%, activity change curves of proteins during fragmentation were plotted (Table 2 and 100). Figure 3 , Figure 3 In the text, a, b, and c correspond to mixtures of FLuc / RLuc, FLuc / EGFP, and FLuc / mCherry, respectively.

[0079] Analysis shows that when FLuc is used as an internal standard, the increasing trend of its activity in the lysed supernatant is basically consistent with the increasing trend of the activity of the three target proteins. This indicates that FLuc plays a good quantitative characterization role in the release process of target proteins and can provide an effective reference for setting ultrasonic disruption parameters under different conditions.

[0080] Table 2. Data on luciferase-assisted quantification of fireflies (n=3)

[0081]

[0082] 3. Sensitivity test of firefly luciferase to ultrasonic disruption power

[0083] In this embodiment, the specific experimental parameters for step 2.6 are as follows: After centrifuging the induced bacterial suspension at 7000g and 4℃ for 2 minutes, the supernatant was discarded, and the bacterial sludge was collected. The sludge was resuspended in 20mL of pre-chilled PBS buffer, and the centrifugation was repeated once more. After discarding the supernatant, the bacterial cells were resuspended in 10mL of pre-chilled PBS. Then, 20μL each of the resuspended bacterial suspensions expressing FLuc and RLuc were added to 10mL of pre-chilled PBS, mixed thoroughly, and placed in a 50mL centrifuge tube in an ice-water mixture. Using a 6mm diameter ultrasonic probe, the pulse disruption conditions were set to "disrupt for 2 seconds, stop for 3 seconds," and disruption was performed using a cell disruptor at power levels of 160W, 240W, and 320W. After centrifugation at 15000g and 4℃ for 15 minutes, the supernatant was collected to detect protein activity.

[0084] The luciferase-expressing bacteria were disrupted using different ultrasonic powers, and the results are shown in Table 3. Figure 4 (The average value corresponding to 100% luciferase activity is shown in the figure.) Figure 4 In the diagram, a, b, and c correspond to 160W, 240W, and 320W, respectively.

[0085] It is evident that high power during the fragmentation process has significant negative effects, and the changes in FLuc activity can indeed reflect the phenomenon of excessively harsh fragmentation conditions, thus providing an important reference for the preservation of protein activity.

[0086] Table 3. Effects of different ultrasonic disruption powers on luciferase activity (n=3)

[0087]

[0088] 4. The effect of adding luciferase on target protein purification

[0089] In this embodiment, the specific experimental parameters for step 2.7 are as follows: After centrifuging the induced bacterial culture at 7000g and 4℃ for 2 minutes, the supernatant was discarded, and the bacterial sludge was collected. The sludge was resuspended in 20mL of pre-chilled PBS buffer, and centrifuged again. The supernatant was discarded, and the bacterial cells were resuspended in 10mL of pre-chilled PBS. Then, 20μL each of the resuspended bacterial cultures expressing FLuc and RLuc were added to 10mL of pre-chilled PBS. After mixing thoroughly, the bacterial culture was placed in a 50mL centrifuge tube in an ice-water mixture. Using a 6mm diameter ultrasonic probe, the pulse disruption conditions were set to "disrupt for 2 seconds, stop for 3 seconds" at 160W power, and the cells were disrupted using a cell disruptor for 20 minutes. The disrupted bacterial suspension was used as the protein solution to be purified.

[0090] Wash the nickel beads three times with PBS (using washing conditions as per the manufacturer's instructions). Add an equal volume of nickel beads to the protein solution (sample to be purified), mix well, and store the sample. Then, vortex at 20 rpm and 4°C for 12 hours, centrifuge at 500g for 3 minutes, and store 50 μL of the supernatant. Wash the nickel beads three more times with PBS, and store 50 μL of the nickel beads. Add 1× SDS loading buffer to each sample, bringing the volume to 100 μL. After sample preparation, take 10 μL for SDS-PAGE (10% separating gel concentration) analysis, using the Cochlear staining method for protein staining.

[0091] In this experiment, FLuc / EGFP or FLuc / mCherry supernatant was mixed at a 2 / 1 (v / v) ratio as the sample to be purified for nickel bead adsorption detection of the tagged protein. Figure 5 The results show that even in samples with an excess of FLuc solution, FLuc is difficult to be adsorbed by nickel beads, and the corresponding bands are basically retained in the supernatant, which is completely opposite to the color development results of EGFP and mCherry.

[0092] Therefore, it can be considered that when FLuc is used as an auxiliary quantitative method for crushing, its impact on the purification process of nickel beads and the purity of the product is negligible.

[0093] 5. The effect of cryopreservation on the effectiveness of auxiliary quantification

[0094] In this embodiment, the specific experimental parameters for step 2.8 are as follows: After inducing expression, the bacterial suspension is centrifuged at 7000g and 4℃ for 2 minutes. The supernatant is discarded, and the bacterial sludge is collected and resuspended in 10mL of PBS containing 30% (v / v) glycerol. The resuspended bacterial suspension is then aliquoted and frozen at -80℃. Subsequently, every 30 days, 1.0mL of bacterial suspension containing both FLuc and RLuc is taken, thawed in a 30℃ water bath, mixed, centrifuged, and resuspended in 10mL of pre-cooled PBS, followed by disruption using 160W power. After centrifugation at 15000g and 4℃ for 15 minutes, the supernatant is used to detect luciferase activity.

[0095] This experiment, following the steps described in 2.8, detected the changes in the activities of two luciferases in cryopreserved bacteria from 0 to 90 days. The results are shown in Table 4 and... Figure 6 As shown, Figure 6 a, b, c, and d correspond to freezing for 0, 30, 60, and 90 days, respectively.

[0096] The results showed that: (1) In the activity curves of the two luciferases, the mean values ​​of the latter three groups of data did not show a decreasing trend in the four consecutive tests, indicating that the activity of the two luciferases did not suffer any detectable loss during the cryopreservation process. (2) In the activity curves of the two luciferases, the slope was the largest in the 0-3 min time period, indicating that the bacteria were most sensitive to ultrasound in the initial stage of disruption. In the four consecutive tests, the value at the 3 min data point fluctuated between 50% and 60% of the mean value of the latter three groups of data, without a significant upward trend. Figure 6 The disruption curve of the fresh bacterial solution shown in figure a is similar to that of... Figure 6 The fragmentation curves of the freeze-thawed bacteria shown in b to 6d also show a similar trend, indicating that a single freeze-thaw cycle has limited impact on the cell wall or cell membrane of E. coli and cannot significantly reduce the bacteria's tolerance to external forces. The fragmentation difficulty of freeze-thawed bacteria is basically the same as that of fresh bacteria.

[0097] Therefore, it can be concluded that after one preparation and freezing, the FLuc expression bacterial suspension can maintain similar detection sensitivity for at least 3 months, and the one freeze-thaw process has no significant impact on the cell disruption tolerance, resulting in excellent signal-to-noise ratio.

[0098] Table 4. Data on luciferase-assisted quantification of fireflies at different freezing times (n=3)

[0099]

[0100]

[0101] Example 2

[0102] The difference between this embodiment and Embodiment 1 is that the final concentration of IPTG added is 50 μM.

[0103] Example 3

[0104] The difference between this embodiment and Embodiment 1 is that the final concentration of IPTG added is 75 μM.

[0105] Example 4

[0106] The difference between this embodiment and Embodiment 1 is that IPTG was added and the culture was continued for 20 hours.

[0107] Example 5

[0108] The difference between this embodiment and Embodiment 1 is that IPTG was added and the culture was continued for 22 hours.

[0109] In summary, this method is an enzyme-assisted quantitative method based on the ultrasonic disruption of Escherichia coli by the release of luciferase activity from fireflies. With its relatively simple operation and stable detection results, it effectively solves the problem of efficiency detection in the ultrasonic disruption process of E. coli. It is suitable for optimizing ultrasonic disruption conditions or for quality monitoring in routine experiments, and provides a scientific basis and new approach for the development of new feed protein resources and livestock and poultry breeding research.

[0110] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase, characterized in that, Escherichia coli expressing firefly luciferase was used as an internal standard and mixed with the target protein expressing bacterial suspension. The cells were then disrupted by sonication. The degree of disruption of the target protein expressing bacterial suspension was quantitatively calculated by measuring the activity of firefly luciferase. The target protein is expressed in the bacterial suspension containing Escherichia coli.

2. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 1, characterized in that, The quantitative method includes the following steps: S1. Induction of target protein expression: Escherichia coli was added to the first culture medium and cultured with shaking once. The culture product was then transferred to the second culture medium and cultured with shaking a second time. IPTG was added to the second culture product and cultured for a second time to obtain the bacterial culture after induction of expression. The first culture medium and the second culture medium are ampicillin-resistant LB medium; S2. Ultrasonic disruption of bacterial cells: Centrifuge the bacterial suspension obtained in step S1 after induction and collect the bacterial sludge, then resuspend it in PBS buffer. Repeat centrifugation and collection of bacterial sludge, then resuspend it in PBS buffer. Mix the internal standard into the bacterial suspension, then perform ultrasonic disruption and centrifugation to obtain the supernatant. S3. Detection of firefly luciferase activity: Take the supernatant obtained in step S2 for luciferase detection.

3. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 2, characterized in that, The Escherichia coli is a glycerol bacterium, and the volume ratio of Escherichia coli to the first culture medium is 1-5:750; the first shaking culture is a shaking culture at 37℃ and 220r / min for 16h.

4. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 2, characterized in that, The volume ratio of the primary culture product to the secondary culture medium is 500 μL: 150 mL; the secondary shaking culture is carried out at 37℃ and 220 r / min until the turbidity of OD650 is 0.5 to 0.

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5. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 2, characterized in that, The final concentration of IPTG is 50–100 μM; the continued culture is carried out at 15 °C and 220 r / min for 20–24 h.

6. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 2, characterized in that, The centrifugation is performed at 6000-8000g and 0-4℃ for 2-5 minutes; the resuspension is performed using PBS buffer pre-cooled at 0-4℃, wherein the pH of the PBS buffer is 7.4 and the concentration is 0.01mol / L.

7. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 2, characterized in that, The volume ratio of the internal standard to the target protein expression bacterial suspension is 1:100 to 1000.

8. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 2, characterized in that, The ultrasonic disruption is carried out in an ice-water mixture incubation process using pulsed disruption. The ultrasonic disruption power is 160–240 W, and the pulse conditions are: disruption for 2 seconds, pause for 3 seconds, and disruption time for 10–20 minutes.

9. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 2, characterized in that, The post-centrifugation is performed at 15000–18000g and 0–4℃ for 10–15 min.

10. The quantitative method for ultrasonic disruption of *E. coli* cells based on firefly luciferase according to claim 2, characterized in that, The luciferase detection was performed using an enzyme-linked immunosorbent assay (ELISA) reader with chemiluminescence detection capability.

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