Method for reducing viscosity of cell disruption liquid

Abstract

The invention belongs to the technical field of biology, and relates to a viscosity reduction preparation of a cell disruption liquid and a method for reducing the viscosity of the cell disruption liquid. The viscosity reduction preparation of the cell disruption liquid comprises escherichia coli thalli for expressing nuclease of non-specific hydrolysis DNA and / or a nuclease crude product prepared from the escherichia coli thalli for expressing nuclease of non-specific hydrolysis DNA; the method for reducing the viscosity of the cell disruption liquid comprises the following steps: mixing the viscosity reduction preparation with the cell disruption liquid to form a cell disruption system, so as to reduce the viscosity of the cell disruption liquid in the cell disruption process; the method is simple, efficient and low in cost.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for reducing the viscosity of cell disruption fluid. Background Art

[0002] Escherichia coli is currently the most widely used prokaryotic expression host. When recombinant proteins are expressed intracellularly in Escherichia coli, cells must first be broken to obtain the recombinant proteins. When Escherichia coli is broken by a milder breaking method such as an enzymatic method, the Escherichia coli chromosomal DNA will be released into the lysate, making the lysate very viscous, affecting subsequent separation steps such as centrifugation. At present, the method of reducing the viscosity of the lysate in the process of breaking Escherichia coli by a milder method usually adds nucleases to hydrolyze the Escherichia coli chromosomal DNA, reducing the viscosity and facilitating separation. The disadvantage is that the cost of nuclease enzymes is relatively high. Another method is to transform genetically engineered bacteria, secrete and express nucleases in the periplasmic space of genetically engineered bacteria, and automatically degrade the released host DNA after cell disruption. This method avoids the high cost of adding exogenous nucleases. However, this method has poor applicability and requires the transformation of Escherichia coli to secrete and express nucleases in the periplasmic space, or to use a transformed strain, which is not suitable for the reduction of viscosity when conventional Escherichia coli strains are broken.

[0003] Therefore, it is necessary to research and develop a low-cost and highly applicable method for reducing the viscosity of Escherichia coli disrupted liquid. Summary of the invention

[0004] One of the purposes of the present invention is to provide a viscosity reducing preparation for cell lysis liquid in view of the deficiencies of the prior art, which can effectively reduce the viscosity of the cell lysis liquid containing the bacteria to be lysised, and is beneficial to the subsequent treatment of the lysis liquid.

[0005] The second object of the present invention is to provide a method for reducing the viscosity of cell disruption fluid, which is simple, convenient, efficient and low-cost.

[0006] To this end, the first aspect of the present invention provides a viscosity-reducing preparation of a cell lysis solution, which comprises Escherichia coli cells expressing a nuclease that non-specifically hydrolyzes DNA and / or a crude nuclease produced by Escherichia coli cells expressing a nuclease that non-specifically hydrolyzes DNA.

[0007] In some embodiments of the present invention, the nuclease that non-specifically hydrolyzes DNA includes one or more of universal nuclease, Staphylococcus aureus nuclease B and deoxyribonuclease I.

[0008] The second aspect of the present invention provides a method for reducing the viscosity of a cell disruption solution, which comprises mixing the viscosity reducing preparation as described in the first aspect of the present invention with a cell disruption solution to form a cell disruption system, so as to reduce the viscosity of the cell disruption solution during the cell disruption process; wherein the cell disruption solution is a mixed solution of a bacterial suspension containing the bacteria to be disrupted after cell disruption.

[0009] In the present invention, the bacteria to be disrupted include Gram-positive bacteria and / or Gram-negative bacteria.

[0010] Preferably, in the cell disruption system, the ratio of the concentration of the viscosity-reducing agent to the concentration of bacteria in the bacterial suspension containing the bacteria to be disrupted is ≥ 1:200,000.

[0011] For example, in some specific examples of the present invention, the bacteria to be disrupted are Gram-negative bacteria, such as Escherichia coli; the method for reducing the viscosity of the cell disruption solution includes adding Escherichia coli cells expressing a nuclease that non-specifically hydrolyzes DNA to a bacterial suspension containing the Escherichia coli to be disrupted to form a cell disruption system. During the disruption process of the Escherichia coli to be disrupted, the Escherichia coli expressing a nuclease that non-specifically hydrolyzes DNA will also be lysed to release the nuclease that non-specifically hydrolyzes DNA, thereby degrading the DNA released in the disruption system and reducing the viscosity of the lysate.

[0012] In some specific examples, the Escherichia coli expressing nucleases that non-specifically hydrolyze DNA include but are not limited to Escherichia coli containing a DNA sequence of a universal nuclease target sequence from Serratia marcescens (as shown in SEQ ID NO.1) designed based on Escherichia coli codon optimization, and Escherichia coli containing a DNA sequence of a Staphylococcus aureus nuclease B (Staphylococcus aureus nucleaseB) target sequence (as shown in SEQ ID NO.2) designed based on Escherichia coli codon optimization.

[0013] In the present invention, the terms "cell disruption solution", "lysis solution" and "cell lysis solution" can be used interchangeably.

[0014] The present invention provides a method for reducing the viscosity of a cell disruption solution. In the method, Escherichia coli cells expressing a nuclease that non-specifically hydrolyzes DNA and cells to be disrupted are two different cells. The present invention reduces the viscosity without directly adding nuclease, and the use of Escherichia coli expressing the nuclease is low-cost, simple and efficient. DETAILED DESCRIPTION

[0015] To make the present invention easy to understand, the present invention is described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0016] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0017] Example

[0018] In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained through commercial channels or conventional methods unless otherwise specified.

[0019] Embodiment 1:

[0020] 1) Obtain E. coli expressing universal nuclease

[0021] A DNA sequence containing the target sequence of the universal nuclease from Serratia marcescens (as shown in SEQ ID NO.1) was designed based on the codon optimization of Escherichia coli, the designed DNA sequence was chemically synthesized, and inserted into pET-28a(+) through the XbaI and EcoRI restriction sites to form a recombinant plasmid pET-nucA.

[0022] The plasmid pET-nucA was transformed into Escherichia coli BL21 (DE3) (Shanghai Shenggong Biotechnology Co., Ltd.), and a single clone colony was picked and inoculated into 3 mL LB culture medium containing kanamycin (50 μg / mL), and cultured at 37°C 200rpm for 16 hours, and then inoculated into 50 mL lactose induction medium (1% peptone, 0.5% yeast powder, 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 25mM Na2HPO4, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM Mg2SO4) containing kanamycin (50 μg / mL) at a 1% inoculation ratio. After culturing at 28°C 200rpm for 24 hours, the cells were centrifuged and collected to obtain Escherichia coli cells expressing the omnipotent nuclease. The cells were washed with water and resuspended, and a portion of the bacterial suspension was frozen and stored at -70°C.

[0023] 2) Viscosity reduction during enzymatic disruption of E. coli

[0024] Inoculate E. coli BL21 (DE3) in LB medium, culture at 37°C 200rpm for 16 hours, then centrifuge and harvest the cells, wash the cells with water and resuspend them, and adjust the cell concentration of the cell suspension to about 20OD OD600. Take 7 1.5mL centrifuge tubes, numbered 0-6, add 500μL of E. coli BL21 (DE3) suspension, then add T4 lysozyme to a final concentration of 1μg / mL, then add E. coli expressing omnipotent nuclease to tubes 0-6 to a final concentration of OD600 of 0OD (tube 0), 0.1OD (tube 1), 0.025OD (tube 2), 0.006OD (tube 3), 0.0015OD (tube 4), 0.0004OD (tube 5), 0.0001OD (tube 6), and mix well.

[0025] The above tubes 0-6 were placed in -70℃ and frozen for 15 minutes, then taken out and thawed at room temperature and continued to be placed. Under the action of freeze-thaw and T4 lysozyme, E. coli was broken, and the intracellular chromosomal DNA was released. Tube 0 began to become sticky within 10 minutes of thawing, and the broken liquid was sticky and stringy; tubes 1, 2, and 3 were placed after thawing without sticky and stringy phenomenon; tube 4 became sticky and stringy within 10 minutes after thawing, and the lysate was no longer sticky 20 minutes after thawing, and the stringy phenomenon of the lysate disappeared; the lysate of tube 5 became sticky after thawing, and the viscosity decreased after thawing and the stringy phenomenon of the lysate disappeared; the lysate of tube 6 became sticky after thawing, and it was no longer sticky after thawing and the stringy phenomenon of the lysate disappeared after thawing, and the lysate was in a sticky state during the placement of tube 0 after thawing. Tubes 0-6 were centrifuged at 12000rpm for 5min. Supernatant and cell fragments were easily obtained from tubes 1-6 by centrifugation, while it was difficult to separate supernatant and cell fragments from tube 0 without Escherichia coli expressing the omnipotent nuclease, indicating that the use of the Escherichia coli expressing the omnipotent nuclease can effectively reduce the viscosity of the lysate.

[0026] Embodiment 2:

[0027] Prepare an Escherichia coli BL21 (DE3) bacterial suspension with an OD600 of about 20 OD according to the method in Example 1. Take 6 1.5 mL centrifuge tubes, numbered 0-5, add 500 μL of Escherichia coli BL21 (DE3) bacterial suspension, and then add T4 lysozyme to a final concentration of 1 μg / mL, and EDTA at pH 8.0 to a final concentration of 2 mM. Take the Escherichia coli bacterial suspension expressing the omnipotent nuclease frozen at -70°C in Example 1, and add it to tubes 0-5 after thawing to a final concentration of OD600 of 0 OD (tube 0), 0.05 OD (tube 1), 0.025 OD (tube 2), 0.0125 OD (tube 3), 0.006 OD (tube 4), 0.003 OD (tube 5), and mix well.

[0028] The above tubes 0-5 were placed in -70℃ freezer for 15 minutes, taken out, and placed at room temperature for thawing and continued to be placed. After the tube 0 was completely thawed, it began to become very viscous and the stringing phenomenon was serious; there was no stringing phenomenon after the 1st and 2nd tubes were thawed; the tube 3 was weakly viscous after thawing, with slight stringing phenomenon, which disappeared after 5 minutes; the lysate of the tube 4 was relatively viscous after thawing, with obvious stringing phenomenon, which disappeared after 20 minutes, and the lysate was no longer viscous; the lysate of the tube 5 was very viscous after thawing, and the stringing phenomenon disappeared after 40 minutes. The tubes 0-5 were centrifuged at 12000rpm for 5 minutes. The supernatant and cell fragments of the tubes 1-5 could be easily obtained by centrifugation, while the supernatant and cell fragments of the tube 0 without the addition of E. coli expressing the omnipotent nuclease were difficult to separate by centrifugation.

[0029] In the presence of 2 mM EDTA, the Escherichia coli expressing the omnipotent nuclease can effectively reduce the viscosity of the lysate.

[0030] Embodiment 3:

[0031] 1) Obtain E. coli expressing Staphylococcus aureus nucleaseB

[0032] A DNA sequence containing the target sequence of Staphylococcus aureus nucleaseB was designed based on codon optimization of Escherichia coli (as shown in SEQ ID NO.2), chemically synthesized, and inserted into pET-28a(+) through XbaI and HindIII restriction sites to form a recombinant plasmid pET-nucB.

[0033] The plasmid pET-nucB was transformed into Escherichia coli BL21 (DE3), and a single clone colony was picked and inoculated into 3 mL LB culture medium containing kanamycin (50 μg / mL), and cultured at 37°C 200 rpm for 16 hours. Then, it was inoculated into 50 mL lactose induction medium (1% peptone, 0.5% yeast powder, 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM Mg2SO4) containing kanamycin (50 μg / mL) at a 1% inoculation ratio. After culturing at 28°C 200 rpm for 24 hours, the cells were collected by centrifugation to obtain Escherichia coli cells expressing Staphylococcus aureus nucleaseB, and the cells were washed with water and resuspended.

[0034] 2) Viscosity reduction during the disruption of E. coli by T4 lysozyme

[0035] Inoculate E. coli BL21 (DE3) in LB medium, culture at 37°C 200rpm for 16 hours, then harvest the cells by centrifugation, wash the cells with water and resuspend them, and adjust the cell concentration of the suspension to about 20OD OD600. Take two 1.5mL centrifuge tubes, numbered 0 and 1, add 500μL of E. coli BL21 (DE3) suspension, then add T4 lysozyme to a final concentration of 1μg / mL, then add E. coli suspension expressing Staphylococcus aureus nucleaseB to the tubes to a final concentration of OD600 of 0OD (tube 0) and 0.1OD (tube 1), and mix well.

[0036] The above tubes 0 and 1 were placed in -70℃ freezer for 15 minutes, taken out, and thawed at room temperature before continuing to stand. After the tube 0 was completely thawed, it began to become very viscous, and the stringing phenomenon was serious; the lysate in the tube 1 was completely thawed and became viscous, and the stringing phenomenon appeared. After being placed for 15 minutes, the stringing phenomenon disappeared, and the lysate was no longer viscous. The tubes 0 and 1 were centrifuged at 12000rpm for 5 minutes. The supernatant and cell fragments of the tube 1 could be easily obtained by centrifugation, while the supernatant and cell fragments of the tube 0 without the addition of E. coli expressing nuclease were difficult to separate, indicating that the use of the E. coli expressing Staphylococcus aureus nucleaseB can effectively reduce the viscosity of the lysate.

[0037] It should be noted that the embodiments described above are only preferred embodiments of the present invention, which are used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A viscosity-reducing preparation for cell lysate, comprising Escherichia coli cells expressing a nuclease that non-specifically hydrolyzes DNA and / or a crude nuclease produced from Escherichia coli cells expressing a nuclease that non-specifically hydrolyzes DNA.

2. The viscosity reducing preparation according to claim 1, characterized in that The nuclease that non-specifically hydrolyzes DNA includes one or more of universal nuclease, Staphylococcus aureus nuclease B and deoxyribonuclease I.

3. A method for reducing the viscosity of a cell disruption solution, comprising mixing the viscosity reducing agent according to claim 1 or 2 with a cell disruption solution to form a cell disruption system, so as to reduce the viscosity of the cell disruption solution during the cell disruption process; wherein: The cell disruption solution is a mixed solution after the bacterial suspension containing the bacteria to be disrupted is subjected to cell disruption.

4. The method according to claim 3, characterized in that The bacteria to be disrupted include Gram-positive bacteria and / or Gram-negative bacteria.

5. The method according to claim 3 or 4, characterized in that: In the cell disruption system, the ratio of the concentration of the viscosity-reducing agent to the bacterial concentration in the bacterial suspension containing the bacteria to be disrupted is ≥ 1:200,000.