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Some tips in the process of protein purification experiments

2021-04-14
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Newbies in protein purification have benefits. Qianchun Biotech will list some of the most commonly used purification processes and conceptual methods. Escherichia coli (E. coli referred to here is a recombinant protein that is not secreted into the culture medium).

About the amount of bacteria

The genetically engineered protein expressed in Escherichia coli is the most convenient raw material for purification personnel, and there are almost no raw material restrictions for the development of purification processes. I often see experimental friends who use a few milliliters of bacterial solution for purification. I am very puzzled about this. Why not do more? Few bacterial cells will cause some difficult to estimate problems in purification, and the repeatability and amplification of the process often have problems. Therefore, to make a good process, it is necessary to ferment and express some bacteria. When our company is doing protein purification, the amount of bacteria used in the initial process is generally about 10g.

Regarding whether the inclusion body is expressed

Regarding this question, I would like to say that whether a genetically engineered protein is expressed in inclusion bodies is not completely accurate in itself. As for the appearance of the crystal particles of the inclusion bodies under the electron microscope, etc., it is meaningless for our purification. I believe that no one who does the purification process will look at this electron microscope and do not care. The basis of our judgment is only SDS-PAGE, and the target protein is in the bacteriolytic precipitation, we think it is the expression of inclusion bodies, but this is a specious conclusion. It looks okay, but it’s actually faulty. The key is what bacteriostatic buffer you are using! Some proteins are in the bacteriostatic precipitation when Buffer A is used to break bacteria, but in the bacteriostatic supernatant when Buffer B is used to break bacteria. The difference between buffer A and B may only be a difference of 1-2 units in pH. So is it expressed in inclusion bodies or in soluble supernatant?

It is said that this problem is mainly because some experimental friends are often very concerned about whether their target protein is expressed in inclusion bodies, and even for inclusion body expression, they use special inclusion body protein purification methods and so on.

In fact, for our company, what we care about is in what buffer system the target protein is soluble and insoluble under what buffer system! Don’t let the concept of inclusion bodies mislead you.

Regarding the amount of expression

We often see in published articles that the expression level of the target protein of my engineered bacteria reaches 30%, 50%, and so on of the total protein of the bacteria. I would say that the author of the article is fooling around. I don’t know how they are quantified, the most used is probably SDS-PAGE scanning analysis. Not to mention that an SDS-PAGE cannot show all the bacterial proteins. The staining method of electrophoresis, the intensity of staining and decolorization, the exposure intensity of the photo, the selection of bands in scanning analysis, etc. all have a huge impact on this percentage. Friends who have done protein QC know how difficult it is to turn 20% of the band into 30%?

Our company’s description of the amount of expression cannot be quantified, but can only be qualitative. Such as: very low, low, medium, high, high, etc. to describe. The difficulty of the purification process is sometimes related to the amount of expression, and purification is often more convenient when the amount of expression is high. Therefore, maximizing the expression of your target protein is not only a matter of upstream and yield of genetic engineering, but also a matter of purification process development.

Broken bacteria

(1) Pre-treatment for bacteriostasis

After the induced expression of the bacterial cell is fermented and centrifuged, it is best to wash it once with PBS buffer or other buffers immediately. Bacteria washing can remove some impurities and metabolites in the culture medium, reducing the impact on subsequent purification. If the cells have been frozen, the frozen cells may be partially broken, so do not wash the cells.

The suspension of a small amount of bacteria can be blown with a 5ml pipette tip, and then magnetically stirred. A dispersing emulsifier can be used to suspend a large amount of bacteria. The amount of bacteriolytic buffer is generally 1:10-1:20, that is, 10-20 ml of bactericidal buffer is used for 1g of wet bacteria.

(2) Selection of bactericidal buffer

The choice of bacteriostatic buffer should be closely related to the subsequent purification method and cannot be a one-size-fits-all solution. Moreover, the bacteriostatic buffer is also related to whether the expression is soluble. For soluble recombinant proteins, the equilibration buffer of the first step of chromatographic purification is generally used as the bactericidal buffer. For insoluble recombinant proteins, the simplest method is to use PBS as the bacteriolytic buffer.

When selecting the bacteriostatic buffer, there is a small tip, adding EDTA, some recombinant proteins are very fragile, and there will be a lot of degradation during ultrasonic bacteriostasis. Note that it is not expression degradation, but degradation during ultrasound. Especially the his-tag fusion protein expressed with pET32 is easy to degrade.

(3) Sterilization method

The commonly used methods for breaking Escherichia coli are probably: repeated freezing and thawing plus lysozyme method, ultrasonic method and pressure homogenization method. Generally speaking, the amount of bacteria processed is in this order. Our company chooses to use powerful ultrasound and pressure homogenization. Ultrasonic method is the first choice for small-scale and medium-scale disruption of bacteria. Choose the corresponding ultrasonic probe, and the amount of bacteria processed at a time is 0.1g-100g. Pressure homogenization is the first choice for medium to large-scale bacteriostasis. The processing capacity is large and fast, and the bacteriostatic effect is good, but the heat is large, and it is used with caution for the temperature-sensitive protein.

Ultrasonic bacteriostasis: Take 10g of wet bacteria as an example, add 100ml of bactericidal buffer, and suspend in a glass beaker with magnetic stirring. Ultrasound in ice water bath. Set ultrasonic time 4s, gap time 4s, ultrasonic power 400-600W, ultrasonic frequency 100-200 times.

Pressure homogenization: Take 200g of wet bacteria as an example, add 3000ml of 4℃ pre-cooled bacteriostatic buffer, and mix thoroughly with a dispersing emulsifier. The homogenization pressure is about 800bar, and the pressure homogenization heats up very much. The effluent should be stirred in an ice water bath and magnetically. After the first homogenization is completed, allow the bacterial liquid to cool sufficiently before performing the second homogenization. If the homogenized bacterial liquid has a relatively high viscosity, it can be sonicated 40 times with an ultrasonic disruptor to break the nucleic acid to reduce the viscosity, so as to facilitate centrifugation and purification.

(4) Centrifuge after bacteriostasis

Centrifuge in a high-speed refrigerated centrifuge, 18000rpm for a 50ml tube, 20min, 4℃; for a 500ml bottle, 10000rpm, 30min, 4℃. The centrifugal effect of a 50ml tube is better than that of a 500ml bottle.

(5) Filter

The soluble protein expressed in the supernatant must be filtered before it can be purified on the column after the supernatant is collected and centrifuged. The filtration generally adopts vacuum negative pressure suction filtration, and you can choose 0.8um membrane or double-layer filter paper to filter. Most chromatographic requirements are that the column solution needs to be filtered through a 0.45um membrane, but in fact, for the supernatant after bacteriostasis or the dissolved supernatant of inclusion bodies, the 0.45um membrane is too difficult to filter and cannot be completed. Therefore, our company adopts double-layer filter paper or 0.8um membrane for filtration.

Inclusion body treatment

(1) Inclusion body washing

What solution is used to wash the inclusion bodies is also related to the subsequent purification method. For example, when followed by ion exchange chromatography, it is better not to add high-concentration salt to the washing solution; when it is followed by Ni column purification, try not to add EDTA to the washing solution. (Ni-TED Purose® 6 Fast Flow can tolerate 100mMEDTA)

pH6.0-pH9.0: High pH makes the inclusion bodies tend to dissolve and removes impurities better, but high pH may degrade the target protein.

10-50mM PB, 10-50mM Tris: maintain a buffer environment, and choose a lower concentration when followed by ion exchange.

0-0.5N NaCl: Salt washing is helpful for the removal of nucleic acids. After salt washing, the ratio of OD280/260 of the inclusion bodies (after dissolution) will increase a lot, that is, a lot of nucleic acid removal, which is beneficial to subsequent purification.

0.5-5mM DTT: Provide a reducing environment, open the disulfide bond in the protein, so that the inclusion bodies tend to dissolve, which is conducive to the removal of contaminated proteins. But the concentration is too high to make the inclusion bodies really dissolve and it is not washing. Subsequent use of Ni-IDA column can not be added, use Ni-NTA column to add less. (Thousand Pure Bio-Ni-TED Purose® 6 Fast Flow can tolerate 20mM DTT)

0-1%beta-ME: Same as DTT, with slightly weaker reduction ability.

0-2N Urea: Denaturant, which is beneficial to the opening of hydrogen bonds, hydrophobic bonds, van der Waals forces in the inclusion body, and is beneficial to washing and removing impurities. The same high concentration will dissolve the inclusion bodies.

0.05-1% Triton X100: non-ionic surfactant.

0-2mM EDTA: chelating agent, remove metal ions, which is conducive to the stability and dissolution of protein.

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