CN110964120A

CN110964120A - Method for purifying prokaryotic expression fusion His tag protein

Info

Publication number: CN110964120A
Application number: CN201911281238.5A
Authority: CN
Inventors: 林金星; 连娜; 王鑫伟
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2020-04-07

Abstract

The invention discloses a method for purifying prokaryotic expression fusion His tag protein, which comprises the following steps: 1) centrifuging the bacterial liquid, and collecting thalli; 2) crushing the heavy suspension thallus; 3) crushing, centrifuging and taking supernatant; preparing a chromatography tube and a filler; 4) combining the supernatant with filler, and cleaning the foreign protein; 5) collecting the target protein; 6) and (5) treating the filler and detecting the protein. The invention provides a method for rapidly purifying fusion His tag protein expressed by pronucleus, which has higher efficiency, and can save a large amount of time cost especially when aiming at the purification of large-system protein.

Description

Method for purifying prokaryotic expression fusion His tag protein

Technical Field

The invention relates to the fields of molecular biology and biochemistry, in particular to a method for purifying prokaryotic expression fusion His tag protein.

Technical Field

The expression of target protein in vitro is an indispensable process for the in vitro research of protein function, and the expression of protein is a molecular biology technology for expressing exogenous gene protein by model organisms such as bacteria, yeast, animal cells or plant cells, and plays a central role in genetic engineering technology.

After the target protein is successfully prokaryotic expressed, the subsequent purification process of the protein is involved. The separation and purification of proteins is widely used in biochemical research applications and is an important operation technology. A typical cell may contain thousands of different proteins, some very abundant, some containing only a few copies. To study a protein of interest, the protein must first be purified from other protein and non-protein molecules. What is needed in the art is a method for purifying proteins, which is simple in operation, short in purification time, and wide in application range.

Disclosure of Invention

In order to solve the problems, the invention provides a method for rapidly purifying prokaryotic expression fusion His tag protein, which can save a large amount of time and improve the working efficiency.

The invention is realized by the following technical scheme:

a method for purifying prokaryotic expression fusion His tag protein comprises the following steps:

(1) collecting the induced thallus, centrifuging for 10min at 4000g, and removing the supernatant;

(2) adding precooled His Wash Buffer into the precipitate to resuspend the thallus, adding a protease inhibitor PMSF with the final concentration of 0.1mM, and fully and uniformly mixing the bacterial liquid;

(3) ultrasonically crushing the resuspended bacterial liquid to a proper crushing state, (BILON 650Y) setting of an ultrasonic crusher: 80%, working time 5sec, intermittent time 7sec, working time 15min, and crushing is repeated for a plurality of times according to the amount of the heavy suspension volume;

(4) centrifuging the crushed bacterial liquid at the temperature of 4 ℃ at 15000g for 70-90 min;

(5) filling Ni-NTA Agarose filler into a chromatography tube, filling Ni-NTA Agarose filler into the chromatography tube, then integrally calling the chromatography tube as a chromatography column, and using ddH with 3 times of the volume of the chromatography column₂Cleaning the filler by O, and then adding His WashBuffer cleaning filler with the volume of 1 time of the chromatographic column for later use at 4 ℃;

(6) putting the centrifuged supernatant in the step (4) into a beaker precooled at 4 ℃ C, adding Ni-NTA Agarose filler cleaned by HisWash Buffer in the step (5), putting the beaker on a magnetic stirrer, and stirring for 30-50min at 4 ℃ with attention paid to the rotation speed not too high;

(7) loading the combined mixed solution into a chromatography tube, washing the column filler with His Wash buffer precooled at 4 ℃ after the bacterial solution completely flows out, so as to wash off non-specifically combined hybrid protein, detecting the content of the hybrid protein with a protein detection solution, and washing until the hybrid protein is completely washed off;

(8) completely removing foreign proteins, sealing the chromatographic column, adding precooled His precipitation Buffer, mixing with filler, and standing for 2 min;

(9) and opening the chromatographic column, collecting the eluent by using a pre-cooled centrifugal tube, and detecting the content of the target protein by using a protein detection solution. According to the experimental requirements, DTT can be supplemented to the collected protein to prevent the protein from forming high polymer, or the protein is dialyzed into working buffer solution, or the protein can be directly subpackaged and stored at-80 ℃;

(10) after the experiment was completed, the Ni-NTA Agarose packing was washed with 1 column volume of 0.5M NaOH, followed by more than 10 volume volumes of ddH₂And cleaning the filler, storing the filler in an ethanol solution with the volume ratio of 20%, and keeping the filler at 4 ℃ for later use.

In the step (1), the amount of the added His Wash Buffer is determined according to the amount of the thallus, and the thallus is usually resuspended to be slightly viscous.

In the step (2), the bacterial liquid is required to be fully resuspended as much as possible, and the bacterial liquid can be fully stirred by a medicine spoon or fully blown by a 5mL liquid transfer device until no obvious macroscopic bacterial block is optimal.

In the step (3), the thallus can be crushed by a high-pressure crusher or an ultrasonic crusher, and it should be noted that an appropriate amplitude transformer is selected by the ultrasonic crusher according to the requirement of the self-crushing volume.

In the step (3), the proper crushing state is that the bacterial liquid is changed from slightly viscous to relatively clear, the color of the bacterial liquid is changed from milky white or deep milky white to slightly reddish and dark, and the surface of the bacterial liquid is slightly layered, namely the bacterial liquid is crushed sufficiently.

In the step (4), the centrifugal machine is precooled to 4 ℃ in advance, the crushed bacteria liquid is centrifuged, the centrifugation time is about 80 minutes, and the precipitated impurities can be fully separated.

In the step (5), a 20mL chromatography tube is selected, and about 1-2mL Ni-NTA Agarose filler is preferably filled. If more protein is purified, a plurality of chromatography tubes can be used simultaneously.

In the step (5), ddH is used immediately after the filler is charged₂The O is washed, preferably with at least 3 column volumes of water. And after washing, adding 1 time of His Wash Buffer of the volume of the chromatographic column for washing, and closing the column for later use when about 3mL of the Wash Buffer remains.

And (3) in the step (6), mixing the centrifuged supernatant with the washed filler in the step (5), re-suspending the filler by using the bacterial liquid supernatant, sucking out the re-suspended filler, and mixing in a container at 4 ℃ for about 30min by using a magnetic stirrer or a mute mixer.

In the step (7), the mixed liquid is poured into a chromatography tube, and in the step, a yellow gun head or a drainage tube can be arranged at the bottom of the chromatography column, so that the liquid outflow speed can be greatly increased. Meanwhile, a 5mL plastic dropper can be used for blowing and beating the heavy suspension filler, so that the phenomenon that the filler is accumulated at the bottom of the tube to slow down the liquid outflow speed is avoided.

In the step (7), after the mixed solution is added, His Wash Buffer can be added to Wash away the mixed protein which can not be combined with the filler, and the washing time is different according to different protein contents in the step. The flow rate of liquid can be increased by always keeping the amount of His Wash Buffer in a relatively large state in the process of washing the foreign protein, and meanwhile, the process of the step can be greatly increased by always blowing and beating the uniformly mixed filler by using a plastic dropper.

In the step (7), in the process of washing away the foreign proteins, whether the foreign proteins are clean or not is monitored at any time, effluent liquid is detected at any time, the effluent liquid is identified by using Coomassie brilliant blue dye solution, 200 mu L of dye solution is added into 40 mu L of effluent liquid to be uniformly mixed, and the color change condition is checked. The control was performed with HisWash Buffer, which proved to be clean if the effluent was in the same color as the His Wash Buffer stain in Coomassie Brilliant blue.

In the step (8), the cleaned filler flows the His Wash Buffer clean, the column is closed, and a small amount of His Elution Buffer is added for resuspension.

In the step (8), the eluted protein is collected and detected by Coomassie brilliant blue, and if the Coomassie blue is changed, His Elution Buffer is added to elute until the eluted protein can not change the Coomassie staining solution into blue.

In the step (10), the filler is timely treated after being used up, and is washed by 0.5M NaOH, so that the filler can be resuspended to enable the treatment to be more sufficient. Washed by water and finally stored in ethanol with the volume ratio of 20 percent at the temperature of 4 ℃ for a long time.

Compared with the prior art, the invention has the following beneficial technical effects:

the method utilizes the premixing of the bacterial liquid and the filler, so that the target protein and the filler can be combined more fully, the column passing stage of the bacterial liquid only needs to be carried out once, and the phenomenon that the target protein can be fully combined with the filler only by directly passing the supernatant through the column for 2-3 times under the condition that the bacterial liquid is not premixed in the prior art is avoided; a yellow gun head or a drainage tube is arranged at the bottom of the chromatographic column, so that the liquid outflow speed can be accelerated, and the time is saved; meanwhile, the method utilizes the disposable plastic dropper to blow and resuspend the filler, so that the liquid flow rate can be greatly accelerated, the foreign protein can be more fully cleaned, and the phenomenon that the liquid flow rate is slowed down due to the fact that the filler is deposited at the bottom of the tube when the bacteria liquid passes through the column and the foreign protein is cleaned is avoided. By adopting the mode, the purification time is greatly reduced, the overall experimental efficiency is improved, the purification efficiency is high, and the used time is short.

The method is not only suitable for purifying a small amount of protein, but also suitable for a purification system with large protein demand, such as research on protein structure, and the like, because the purification of a plurality of chromatography tubes can be carried out simultaneously in the method, the purification efficiency is improved, and the single purification amount can reach 10L of bacterial liquid under the condition of a common laboratory; in addition, the method of the invention needs simple reagents, only needs two buffers of His Wash Buffer and His Elution Buffer in the whole process, and is simple and easy to prepare.

Detailed Description

The present invention will be described in further detail with reference to specific examples, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The purification method and process of the present invention will be further described in detail below by taking as an example the purification of His-PtrMDP25 protein expressed by E.coli DE3 competent cells.

Example 1: in vitro prokaryotic expression purification (large system) of poplar His-PtrMDP25 protein, which comprises the following specific steps:

(1) the induced bacterial solution (15 bottles, 600mL each bottle, 9L in total) was centrifuged, and the cells were collected under 4000g for 10 min.

(2) The collected mycelia were resuspended to 300mL with His Wash Buffer (His tag washing Buffer), placed in a 500mL glass beaker, resuspended on ice, sufficiently crushed with a long handled spatula, stirred until no clumpy and flocculent mycelia were evident, and 3mL of 0.1M PMSF (phenylmethylsulfonyl fluoride) was added.

(3) Placing the glass beaker in an ice box, crushing with an ultrasonic crusher (BILON 650Y, No. 10 horn), with 80% power, 5sec of working time, 7sec of intermittent time, and ultrasonic crushing for 15 min; and (4) checking whether ice is enough and the position of the amplitude transformer is proper after each ultrasonic treatment, and lightly shaking the beaker to fully mix the bacterial liquid. The ultrasonication was repeated 3 times.

(4) And centrifuging the crushed bacterial liquid at 4 ℃ for 90 min.

(5) And (5) preparing a chromatography tube and a filler while centrifuging the bacterial liquid. The 20mL chromatography tube is cleaned, 3mL Ni-NTA Agarose (nickel chelate affinity chromatography column) filler is added into each tube, and a small section of the upper part of the yellow gun head is cut off and arranged at the lower end of the chromatography tube. The column was washed with 3 column volumes of about 60mL ddH2O, followed by 1 column volume of His Wash Buffer, which was blocked when 3mL remained and kept at 4 ℃.

(6) Pouring the centrifuged bacterial liquid into a precooled 500mL beaker, adding the prepared filler into the beaker after the centrifuged supernatant is resuspended, placing the beaker on a magnetic stirrer in a refrigerator at 4 ℃ for fully mixing for 30min, and paying attention to the fact that the rotating speed is not too high.

(7) The chromatography tube is hung in a refrigerator at 4 ℃, mixed supernatant is poured into the chromatography tube, a yellow gun head is installed at the bottom of the chromatography column, His Wash Buffer can be added immediately after the mixed liquid is poured, and the filler is continuously blown by a plastic dropper in the process to accelerate the outflow speed. The effluent liquid is detected at any time in the process of flushing the filler, 40 mu L of the effluent liquid is added into 200 mu L of Coomassie brilliant blue dye liquid for detection, and His Wash Buffer is taken as a control.

(8) After the His Wash Buffer is drained, the columns are closed, 5mL of His Elutionbuffer (His tag Elution Buffer) is added into each chromatographic column for resuspension, the mixture is kept still for 2 minutes, 10mL of centrifuge tube is used below each chromatographic column for collecting effluent, after 5mL of effluent is drained, 40 mu L of eluent at the tube opening of the chromatographic column is absorbed for detection, if the detection liquid turns blue, which indicates that the protein is not eluted, the 5mL of His Elutionbuffer is continuously added for Elution until the eluent at the tube opening is detected to be not turned blue.

(9) DTT (dithiothreitol) was added to the eluted protein to a final concentration of 5mM to prevent the protein from forming a high polymer. 20 mu L of protein is taken for polyacrylamide gel electrophoresis detection.

(10) After the use of the chromatographic columns, 5mL of 0.5M NaOH was added to each chromatographic column to resuspend the filler, after draining, the filter was washed with 3 column volumes of ddH2O, and finally with 1 column volume of 20% ethanol, wherein 20% ethanol was 20% by volume, and the remaining 3mL of the filter was resuspended in the filler and stored at 4 ℃.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for purifying prokaryotic expression fusion His tag protein comprises the following steps:

(1) centrifuging the induced bacterial liquid, discarding the supernatant, and collecting the thallus;

(2) adding the collected thalli into a precooled His Wash Buffer for resuspension, and carrying out ultrasonic crushing on the resuspended bacterial liquid to a proper crushing state;

(3) centrifuging the crushed bacterial liquid, and taking supernatant; meanwhile, preparing a chromatographic tube and a filler, filling the Ni-NTA Agarose filler into the chromatographic tube to form a chromatographic column, cleaning the filler by ddH2O with the volume of 3 times of the volume of the chromatographic column, and then adding His Wash Buffer with the volume of 1 time of the volume of the chromatographic column to clean the filler;

(4) combining the supernatant obtained in the step (3) with the filler prepared in the step (3), loading the combined mixed solution into a chromatography tube, washing the filler in the chromatography tube by using precooled His Wash Buffer after the bacterial solution completely flows out so as to Wash away the foreign proteins, detecting the content of the foreign proteins by using a protein detection solution, and washing until the foreign proteins are completely washed away; when the mixed liquid is poured into the chromatography tube, a yellow gun head or a drainage tube is arranged at the bottom of the chromatography column, so that the liquid outflow speed can be greatly accelerated, and meanwhile, a plastic dropper is adopted to blow and beat the heavy suspension filler, so that the phenomenon that the filler is accumulated at the bottom of the chromatography tube to slow down the liquid outflow speed is avoided;

(5) collecting the target protein: completely removing foreign proteins, sealing the chromatographic column, adding precooled His ElutionBuffer, and mixing with the filler; opening the chromatographic column, collecting the eluent by using a precooled centrifugal tube, and detecting the content of the target protein by using a protein detection solution;

(6) and (5) treating the filler and detecting the protein.

2. The method of claim 1, wherein: in the step (2), the bacterial liquid is required to be fully resuspended as much as possible, and the bacterial liquid is fully stirred by a medicine spoon or fully blown by a 5mL liquid transfer device until no obvious macroscopic bacterial block exists.

3. The method of claim 1, wherein: in the step (2), the proper crushing state is that the bacterial liquid is changed from sticky to clear, the color of the bacterial liquid is changed from milky white or deep milky white to red and dark, and the surface of the bacterial liquid is layered, namely the bacterial liquid is crushed sufficiently.

4. The method of claim 1, wherein: in the step (3), a 20mL chromatography tube is selected, and about 2mL of Ni-NTA Agarose filler is filled; if more protein is purified, multiple chromatography tubes are used simultaneously.

5. The method of claim 1, wherein: and (4) mixing the centrifuged supernatant with the washed filler, re-suspending the filler with the supernatant of the bacterial liquid, sucking out, and mixing in a container at 4 ℃, wherein a magnetic stirrer or a silent mixer can be used in the process.

6. The method of claim 1, wherein: in the step (4), in the process of washing away the foreign proteins, detecting effluent liquid at any time, identifying by using Coomassie brilliant blue dye liquor, adding the dye liquor into the effluent liquid, mixing uniformly and checking the color change condition; and performing control by using His Wash Buffer, and if the effluent and the His Wash Buffer dye solution have consistent colors in Coomassie brilliant blue, the effluent is proved to be washed clean.

7. The method of claim 1, wherein: in the step (5), the eluted protein is collected and detected by Coomassie brilliant blue, and if the Coomassie blue is changed, His Elution Buffer is added continuously until the eluted protein can not change the Coomassie staining solution into blue.

8. The method of claim 1, wherein: in the step (6), the solution is washed by 0.5M NaOH and is resuspended to ensure that the treatment is more sufficient; washed with water and finally stored in ethanol with the volume ratio of 20 percent for a long time in an environment with the temperature of 4 ℃.