Experimental methods of inclusion bodies, definition and separation methods of inclusion bodies

Inclusion bodies are host cells that express foreign genes, which can be prokaryotic cells, such as Escherichia coli; It can also be eukaryotic cells like yeast cells, mammalian cells, etc. Inclusions are proteins that often form a protein-like lesion structure in the host cell during the proliferation of the virus, which can be seen under a light microscope. Most of them are round, oval or amorphous.

It is generally formed by aggregation of intact viral particles or unassembled viral subunits; A few are the products of the host cell's response to viral infection and do not contain virions.

Separation: Inclusion bodies are located in the cytoplasm or peripheral of the cell, and the cell membrane must be disrupted to release the inclusion bodies. The inclusion bodies in the solution should also be separated from the dissolved components in the fragmentation solution and other insoluble components such as cell debris, cell membranes, and ribosomes.

The first step of extraction is to cool the fermentation broth, collect the cells by centrifugation or cross-flow filtration, and the cell pellet is ...... containing a set concentration

You can do protein concentration first, if you think it is necessary, but I personally never concentrate before the column, why do you need one more step, chromatography will have a concentration effect. 2.The protein concentration method is relatively simple to do in the laboratory or PEG, and only one dialysis bag is required.

But I personally like to overthink, I'm sorry. Precipitating with (NH4)2SO4 or acetone is not recommended. 3.

Regardless of the method of concentration, if the concentration is high and the protein is precipitated in large quantities, then sorry, please re-optimize your refolding protocol. But please also avoid over-concentrated protein, if a protein is stable below 2mg ml, then do not concentrate to 3mg 8m urea will generally not affect your hanging column. 5.

When protein is concentrated by embedding dialysis tubing with PEG dry powder, all small molecules (as long as the molecular weight is less than the molecular weight of the dialysis tubing retained) can enter and exit freely. In general, I have seen more dialysis bags with about 10kd in the laboratory, and I think you should also use this, so please don't use PEG6000 dry powder, you are not doing concentration, you are doing PEG precipitation, because your PEG is running into the dialysis bag.

The cells (or bacteria) can be disrupted by ultrasonic disruption to release the inclusion body, and the fragmentation effect can be detected by microscope; The supernatant solution is then centrifuged, and in general, the recombinant protein of interest is present in the supernatant and can be detected by electrophoresis. The protein is then isolated and purified by appropriate methods, including the refolding of the protein. Recombinant protein separation and purification methods include zeosieve chromatography, ion exchange, hydrophobic chromatography, affinity chromatography and other methods.

In vitro, the expression of exogenous prokaryotic cells, especially Enterobacter bacteria, is efficiently expressed, and the high-density, soluble protein particles are encapsulated by membranes, and the high-refractive region is significantly different from the cytoplasm by microscopic observation.

Prokaryotic protein expression purification is easy to form inclusion bodies, and at this stage, two versions can be used, one is prevention, and the other is resumption.

First of all, "prevention" refers to the optimization of the expression environment before the overexpression of the protein to form an inclusion body, so as to reduce the rate of recombinant protein synthesis. For example: codon optimization, selection of expression temperature, co-expression with chaperone or folded enzyme, etc.; And the refolding of proteins refers to:

When the denaturation conditions are not drastic and the internal structure of the denatured protein does not change much, the denaturation factor is removed, and the denatured protein can restore its natural conformation and biological activity under appropriate conditions.

These two methods are more troublesome to say, and there are many knowledge points and concepts involved, so it is better to read some information first.

Depending on the purpose of the desired protein, if the activity is measured, the vector or strain must be changed, or even the induction conditions are made so that the amount of inclusion is as small as possible; Or denaturation and purification, and then renaturation, but the protein activity of this method will be refolded will lose a lot, basically the activity is lost, and the possibility of activity is not ruled out; If it is used for antibodies or other activities, it does not need to be experimented, just direct denaturation and purification.