Escherichia coli crushing method

Abstract

The invention relates to the technical field of biology, in particular to a crushing method of escherichia coli applied to protein expression. According to the method disclosed by the invention, the escherichia coli is crushed by combining lysozyme and high-pressure homogenization, so that the thallus crushing degree is effectively improved, and the protein concentration and purity are effectively improved. In addition, the use pressure of the homogenizer is effectively reduced, and the service life of machine accessories is prolonged. In addition, the lysozyme is kept active by adopting calcium ions, the use amount of the lysozyme is reduced, and the production cost is reduced.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for disrupting Escherichia coli used for protein expression. Background Art

[0002] In the biopharmaceutical field, proteins with specific functions are highly sought after, such as recombinant insulin for diabetes treatment and interferon for antiviral applications. Escherichia coli, due to its rapid growth and ease of cultivation, is a common host for the expression and production of these proteins. However, after synthesis within E. coli cells, these recombinant proteins are encapsulated within the cellular structure, necessitating cell disruption to release the target proteins. This is essential for obtaining these pharmaceutically valuable products. From a cellular structural perspective, E. coli is a Gram-negative bacterium with a cell wall composed of an outer lipopolysaccharide layer (approximately 8-10 nm thick, consisting of lipid A, core polysaccharide, and O-specific side chains) and an inner peptidoglycan layer. This complex structure makes the cells relatively tough, making cell disruption more challenging. However, cell disruption is a crucial step necessary for subsequent isolation and purification of the recombinant proteins, enabling their industrial production and application. From a production process perspective, cell disruption of recombinant E. coli is a crucial step in the protein production process, directly impacting the efficiency and quality of the target product. If cell disruption is ineffective, protein release is incomplete, reducing the yield of subsequent separation and purification. Using an inappropriate disruption method can also lead to protein denaturation and inactivation, rendering the entire production process meaningless. Therefore, finding a simple, efficient method for disrupting E. coli cells that maintains protein activity is crucial to the development of the biopharmaceutical industry and is crucial to the production of qualified, effective pharmaceutical protein products to meet patient treatment needs.

[0003] There are various methods for disrupting E. coli, each with its own characteristics, including physical, chemical, and biological methods. Lysozyme specifically recognizes and hydrolyzes the β-1,4 glycosidic bonds in the peptidoglycan layer of the E. coli cell wall, causing the cells to become hypertonic, absorbing water and swelling until they rupture, thereby releasing the target product within the cells.

[0004] This method is highly specific, targeting only the peptidoglycan structure of the bacterial cell wall. It effectively protects target products within the cell, such as proteins and nucleic acids, and maximizes their structural integrity and biological activity. However, its high cost, long duration of action, susceptibility to environmental factors, and uneven cell wall-breaking effects on bacteria in different states make it difficult to scale up industrially.

[0005] At the same time, high-pressure homogenizers use high pressure to force bacterial liquid through narrow gaps, generating high-speed flow and strong shear forces that stretch, twist, and break cells. Their high disruption efficiency allows for large-scale, continuous operation, resulting in relatively uniform cell disruption and better preservation of protein activity, facilitating subsequent protein separation and purification. However, these devices are expensive, require high maintenance, and are subject to high operating pressure, presenting safety risks. They may not be effective for certain cell structures and may cause localized overheating, requiring cooling to prevent protein denaturation. Summary of the Invention

[0006] In view of this, the present application provides a method for disrupting Escherichia coli for protein expression, with the aim of providing a bacterial disruption method that is fast, efficient, easy to operate, and can be used for large-scale industrial production.

[0007] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0008] The present application provides a method for disrupting Escherichia coli, comprising: disrupting the bacteria based on lysozyme coordinated by calcium ions.

[0009] In some specific embodiments of the present application, the above-mentioned fragmentation method includes: mixing a mixed solution containing lysozyme and calcium ions with Escherichia coli, and then performing high-pressure homogenization to complete the fragmentation.

[0010] In some specific embodiments of the present application, in the above-mentioned fragmentation method, every 500 mL of the mixed solution contains 100 g of Escherichia coli, 0.3 g, 0.4 g, 0.5 g or 0.6 g of lysozyme, and a final concentration of 1.0, 1.5, or 2.0 M calcium ions.

[0011] In some specific embodiments of the present application, the mixed solution of the above-mentioned fragmentation method further comprises PBS.

[0012] In some specific embodiments of the present application, the pressure of the high-pressure homogenization in the above-mentioned crushing method is 400 bar, 500 bar or 600 bar.

[0013] In some specific embodiments of the present application, the high-pressure homogenization in the above-mentioned crushing method is performed 1 time, 2 times, 3 times, 4 times, 5 times or 6 times.

[0014] In some specific embodiments of the present application, the above-mentioned crushing method comprises the following steps:

[0015] 100 g of Escherichia coli was mixed with 500 mL of buffer, and then mixed evenly with 0.3 g, 0.4 g, 0.5 g, or 0.6 g of lysozyme and 0.25, 0.5, or 1.0 mol of calcium salt to obtain a mixed solution;

[0016] The mixed liquid is subjected to high-pressure homogenization at a pressure of 400 bar, 500 bar or 600 bar;

[0017] Collect the bacterial liquid after high-pressure homogenization, adjust the pH to 3.5-4.0, centrifuge, obtain the supernatant, adjust the pH to neutral, and complete the disruption.

[0018] In some specific embodiments of the present application, the buffer solution in the above-mentioned disruption method is PBS.

[0019] In some specific embodiments of the present application, the calcium salt in the above-mentioned crushing method is CaSO4·2H2O.

[0020] In some specific embodiments of the present application, the lysozyme in the above-mentioned crushing method is 0.4 g, the CaSO4·2H2O is 1 mol, and the pressure is 600 bar.

[0021] In some specific embodiments of the present application, the above-mentioned fragmentation method further includes a step of purifying the protein, including: saturated adsorption of the supernatant after pH adjustment with a filler, balancing, and eluting with a buffer to obtain a purified protein.

[0022] In some specific embodiments of the present application, the filler in the above-mentioned fragmentation method is washed and balanced with purified water before protein purification.

[0023] In some specific embodiments of the present application, the filler in the above-mentioned crushing method is a hydrophobic filler.

[0024] The present invention has the following beneficial effects:

[0025] The combined crushing method of Escherichia coli provided by the present invention can effectively improve the degree of bacterial crushing, with a crushing rate of up to 98.8%, and the protein concentration and purity of the combined crushing method are effectively improved. The crushing pressure of existing high-pressure homogenizers is generally 800-1000 bar, while the crushing pressure of the present invention is 400-600 bar, which effectively reduces the operating pressure of the homogenizer and extends the life of the machine accessories. Moreover, Ca 2+ The activity of lysozyme is maintained, the dosage of lysozyme is reduced, and the production cost is lowered. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0027] Figure 1 The HPLC graph of experimental group 10 using lysozyme in combination with a high-pressure homogenizer is shown;

[0028] Figure 2The HPLC graph of experimental group 11 using lysozyme and high-pressure homogenizer in combination is shown;

[0029] Figure 3 The HPLC graph of experimental group 12 using lysozyme and high-pressure homogenizer in combination is shown. DETAILED DESCRIPTION

[0030] The present application discloses a method for crushing Escherichia coli applied to protein expression. Those skilled in the art can refer to the contents of this application and appropriately improve the process parameters to achieve the above. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of the present invention.

[0031] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0032] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0033] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0034] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and does not limit the scope of the present application. No language in this specification should be construed as indicating any non-claimed element is essential to the practice of the present application.

[0035] In addition, the numerical ranges and parameters used to define this application are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0036] In some embodiments, the bacterial disruption method of the present application uses Ca 2+ The solution is broken.

[0037] In specific implementation, the amount of lysozyme added is 3-6 mg / g of bacteria, and the Ca 2+ The amount added is the final concentration of 1~2 M.

[0038] In one example, lysozyme and Ca were added 2+ The resulting bacterial solution was stirred at 60°C for 1 h.

[0039] In one example, Ca 2+ In the form of Ca 2+ Soluble inorganic salts and their crystalline hydrates.

[0040] In some embodiments, the solution after bacterial disruption is further disrupted 3 to 4 times using a high-pressure homogenizer, and the homogenization pressure is 400 to 600 bar.

[0041] In one example, the bacterial disruption method of the present application includes the following steps:

[0042] Take an appropriate amount of bacteria, prepare a PBS solution of the bacteria, and add appropriate amounts of lysozyme and Ca in proportion. 2+ Stir evenly to obtain a mixed solution;

[0043] The mixed liquid is added to a high-pressure homogenizer and crushed at a homogenization pressure of 400-600 bar;

[0044] The bacterial liquid after high-pressure homogenization was collected, the pH was adjusted to 3.5-4.0, and the supernatant was obtained by centrifugation. The pH was adjusted to neutral to obtain the broken product.

[0045] In some embodiments, a purification step is further included, which may include: washing the supernatant after centrifugation with purified water using a hydrophobic filler, saturating the supernatant for 1 hour after equilibration, eluting with 1× PBS for 15 minutes after re-equilibration, and collecting the eluate to obtain the purified protein.

[0046] In the present application, when lysozyme and high-pressure homogenizer are used in conjunction, lysozyme first destroys the cell wall, makes the cell structure become fragile, and then is processed by high-pressure homogenizer, can more efficiently break Escherichia coli, improves the crushing rate. Meanwhile, lysozyme acts gently, can reduce the damage to protein, and high-pressure homogenizer can complete crushing under relatively low pressure, reduces the influence on protein activity due to high pressure, high temperature, etc., is conducive to the separation and purification of subsequent protein, reduces purification difficulty and cost. Certain metal ions, such as calcium ions, can interact with certain amino acid residues in the lysozyme molecule, stabilize the spatial structure of the enzyme, make the active center of the enzyme better maintain its specific conformation, thereby improving the affinity of the enzyme to the substrate, enhancing the vitality of lysozyme.

[0047] The "breakage rate" referred to in this application refers to the proportion of breakage, which is the percentage of the number of destroyed bacteria observed under a microscope to the total number of bacteria.

[0048] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this application are all common commercial products and can be purchased from the market.

[0049] The present invention will be further described below with reference to the embodiments.

[0050] Example 1: Lysozyme method for disrupting Escherichia coli

[0051] E. coli cells were collected by centrifugation at room temperature. 100 g of cells were weighed into four beakers, 500 mL of 1× PBS was added, and the cells were thoroughly stirred. 400 mg of lysozyme (purchased from McLean, Cat. No. L799345, activity ≥20,000 units / g; source: egg white) was added to each beaker. 0 g, 86.09 g, 129.135 g, and 172.18 g of CaSO₄·2H₂O were added to the four beakers, respectively. Heat in a 60°C waterbath with stirring for 1 hour. The pH was adjusted to 3.5–4.0, and the cells were centrifuged at 7500 rpm for 15 minutes. The supernatant was then adjusted to a pH of 7.2–7.5. Microscopic examination revealed cell disruption rates of 80%, 82.5%, 83.2%, and 84.5%, respectively. The supernatants were collected for subsequent samples.

[0052] Example 2: High-pressure homogenizer method for breaking up Escherichia coli

[0053] Transfer 100 g of bacterial cells to three beakers or conical flasks, add 1000 mL of 1× PBS, and stir thoroughly with a glass rod. Pour the mixed bacterial slurry into the collection cup of a homogenizer. Start the homogenizer and tighten the pressure valve to 400 bar, 500 bar, and 600 bar, respectively. Repeat homogenization 3–4 times. Collect the solution flowing out of the outlet and adjust the pH to 3.5–4.0. Centrifuge at 7500 rpm for 10 minutes, and adjust the pH of the supernatant to 7.2–7.5. Microscopic examination revealed bacterial cell disruption rates of 81.6%, 82.9%, and 84.8%, respectively. Collect the supernatant for subsequent samples.

[0054] Example 3: Lysozyme combined with high-pressure homogenizer to disrupt Escherichia coli

[0055] E. coli cells were collected, 100 g of cells were weighed, and a 10-fold volume of 1× PBS (500 mL) was added. The experimental design was performed according to the following orthogonal experimental table. An appropriate amount of lysozyme was added to the E. coli containing PBS, stirred at 60°C for 1 hour, and then placed in the collection cup of a homogenizer. The homogenizer was started and the pressure valve was tightened to the appropriate pressure. The solution flowing out of the outlet was collected and the pH was adjusted to 3.5-4.0. The solution was centrifuged at 7500 rpm for 10 minutes, and the pH of the supernatant was further adjusted to 7.2-7.5. Microscopic examination revealed that the highest bacterial disruption rate, reaching 98.8%, was achieved when 400 mg of lysozyme was added, 172.18 g of CaSO₄·2H₂O (g), and the homogenizer pressure was 600 bar. The supernatant was collected for subsequent samples.

[0056] Table 1: Group design table for combined use of lysozyme and high-pressure homogenizer

[0057]

[0058] Example 4: Detection of protein concentration by nucleic acid detector and protein purity by high performance liquid chromatography

[0059] Place 1 mL of filler in a chromatography column and rinse the column with 10 column volumes of purified water to completely remove the preservative solution from the filler. After equilibration with ammonium sulfate buffer, add sufficient supernatant to saturate the filler for 1 hour. After re-equilibration, add 8 column volumes of 1× PBS and elute at room temperature for 15 minutes. Collect the eluate for purity analysis.

[0060] Collect the eluate, dilute it 10 times with PBS, and use PBS as blank to measure OD 280 , calculate the sample concentration. The protein concentration of experimental group 10-12 is the highest.

[0061] The three groups with the highest concentrations were subjected to liquid phase purity analysis. The mobile phase A of the HPLC was an aqueous solution containing 0.1% trifluoroacetic acid, and the mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. The column oven temperature was set to 40°C. Mobile phases A and B were placed in the designated positions and vented. After completion, the total flow rate was set to 0.8 mL / min, the automatic injection volume was 20 µL, the detection wavelength was 220 nm, the maximum pump pressure was 20 MPa, and the chromatographic elution conditions were set according to Table 2. The peak area and the total chromatographic peak area excluding the solvent peak on the chromatogram were measured, and the sample purity was calculated by the area normalization method. The results are shown in Table 2. Figures 1 to 3 The protein purity of experimental group 12 was as high as 97.8%.

[0062] Table 2: HPLC procedure

[0063]

[0064] Table 3: Bacterial disruption and protein concentration using lysozyme and high-pressure homogenizer

[0065]

[0066] Table 4: Liquid phase analysis - protein purity

[0067]

[0068] The present invention provides a novel Escherichia coli disruption method, which significantly improves the bacterial cell disruption efficiency and provides a new idea for industrial recombinant protein purification.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of this application.

Claims

1. A method for crushing Escherichia coli, characterized in that: include: The fragmentation is based on the calcium ion coordinated lysozyme.

2. The crushing method according to claim 1, wherein: include: The mixture containing lysozyme and calcium ions is mixed with Escherichia coli and then subjected to high-pressure homogenization to complete the disruption.

3. The crushing method according to claim 2, characterized in that: Each 500 mL of the mixed solution contains 100 g of Escherichia coli, 0.3-0.6 g of lysozyme, and a final concentration of 1-2 M calcium ions.

4. The crushing method according to claim 2, characterized in that: The mixture also contains PBS.

5. The crushing method according to claim 2, characterized in that: The pressure of the high-pressure homogenization is 400-600 bar.

6. The crushing method according to claim 2, characterized in that: The high pressure homogenization is performed 3 to 4 times.

7. The crushing method according to claim 1, wherein: The following steps are involved: Mix 100 g of Escherichia coli with 500 mL of buffer, and then mix evenly with 0.3-0.6 g of lysozyme and 0.5-1 mol of calcium salt to obtain a mixed solution; The mixed liquid is homogenized under high pressure at a pressure of 400-600 bar; Collect the bacterial liquid after high-pressure homogenization, adjust the pH to 3.5-4.0, centrifuge, obtain the supernatant, adjust the pH to neutral, and complete the disruption.

8. The crushing method according to claim 7, characterized in that: The buffer solution is PBS.

9. The crushing method according to claim 7, characterized in that: The calcium salt is CaSO4·2H2O.

10. The crushing method according to claim 9, characterized in that: The amount of lysozyme is 0.4 g, the amount of CaSO 4 ·2H 2 O is 1 mol, and the pressure is 600 bar.

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