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Inclusion body protein purification and renaturation

2020-06-19
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Inclusion bodies: Under certain growth conditions, genetically engineered bacteria can accumulate certain special biological macromolecules, which are densely concentrated in cells, or enveloped by a membrane or form a bare membrane-free structure. This water-insoluble structure is called inclusion body. (Inclusion Bodies, IB).
The composition and characteristics of inclusion bodies
Generally contains more than 50% recombinant protein, the rest are ribosomal elements, RNA polymerase, outer membrane proteins, etc., circular or nicked plasmid DNA, and liposomes, lipopolysaccharides, etc., the size is 0.5-1um, insoluble in water , Only soluble in denaturants such as urea, guanidine hydrochloride, etc.
Inclusion bodies have nothing to do with the type of protein and expression system, only the result of protein overexpression.

Main causes of inclusion bodies

  1. The expression level is too high: the reason may be that the synthesis speed is too fast, so that there is not enough time to fold, the disulfide bonds cannot be paired correctly, too many non-specific bindings between proteins, and the protein cannot achieve sufficient solubility, etc. .

  2. Amino acid composition of recombinant protein: Generally speaking, the more sulfur-containing amino acids, the easier it is to form inclusion bodies

  3. The environment of the recombinant protein: When the fermentation temperature is high (37-42℃) or the intracellular pH is close to the isoelectric point of the protein, inclusion bodies are easily formed.

  4. The recombinant protein is a heterologous protein of Escherichia coli. Due to the lack of enzymes required for post-translational modification in eukaryotes, a large amount of intermediates are accumulated, and inclusion bodies precipitate easily. Co-expression of molecular chaperones is used to increase the proportion of soluble proteins.
    Basic steps for isolation and purification of bacterial inclusion body proteins
           Broken cell
           (Disruption of cell)
           ↓
             Separate inclusion bodies
     (Seperation of inclusion body)
            ↓
             Dissolve inclusion bodies
     (Dissolve inclusion body)
               ↓
               Protein product conformation restoration, etc. (Recovery of target protein conformation)

Basic steps for purification and renaturation of inclusion body proteins

Inclusion body protein purification and renaturation steps

Protein renaturation is the most critical and complex issue in recombinant protein purification. The nature of the protein is different and the environment is different, which makes the renaturation conditions very different. Any protein has an optimal renaturation condition. Only when a suitable renaturation buffer is selected to make the protein fold correctly, can further chromatographic separation be successfully completed.

Refolding characteristics of Medicilon inclusion body protein

  •   High recovery rate of active protein

  •   The correct renaturation product is easily separated from the wrong folded protein.

  •   After folding and refolding, you should get a higher concentration of protein products

  •   The renaturation process takes less time


Common methods of refolding

  1. Dilution renaturation: directly add water or renaturation buffer, the disadvantage is that the volume increases greatly and the subsequent treatment is difficult.
    Dilute protein concentration: high concentration is easy to form aggregates (lower renaturation yield). Sometimes less than 0.01mg/mL.
    Pulsed flow refolding: added to the buffer in batches to keep the folded intermediate at a low level. Example: At a final concentration of 5-10 mg/mL, the refolding yield of lysozyme can reach more than 80%.

  2. Refolding of dialysis: The advantage is that it does not increase the volume. By gradually reducing the concentration of the external permeate to control the removal rate of the denaturant, the speed is slow. It is not suitable for large-scale operations and cannot be applied to production scale.

  3. Ultrafiltration renaturation: select a membrane with a suitable molecular weight retention to allow denaturant to pass through the membrane and prevent protein from passing through. It is more used in production and has a larger scale. The disadvantage is that it is not suitable for a small sample size, and some proteins may be irreversibly denatured during the ultrafiltration process (proteins accumulate on the membrane).

  4. Chromatographic renaturation: auxiliary means with separation.
    4.1 Refolding of gel filtration: Except for the mass transfer and diffusion of protein in the colloidal particles, no other effect occurs between the protein and the medium. This method can play a certain role in inhibiting agglutination, and the removal of denaturants such as urea in gel filtration is relatively slow, which is beneficial to the renaturation of some proteins.
    4.2 Adsorption chromatography renaturation: ion exchange, hydrophobic chromatography, affinity chromatography and expanded bed chromatography all belong to adsorption chromatography. The basic renaturation principle is that after the chromatography column is equilibrated, the denatured protein is loaded and adsorbed on the gel medium, and then the unadsorbed denaturant and miscellaneous protein are washed away with washing buffer, and finally the adsorbed The protein elutes and renaturation is completed during the elution.

  5. Molecular chaperones: mainly including thioredoxin disulfide bond isomerase, peptidyl-colyl cis-trans isomerase, etc. Molecular chaperones and folding enzymes can not only regulate the balance of protein folding and aggregation processes in cells, but also promote the folding and refolding of proteins in vitro.
    In vivo renaturation mainly involves adding genes of molecular chaperone for co-expression during the cultivation of engineering bacteria; in vitro renaturation mainly dissolves inclusion bodies in genetically engineered bacteria, and then adding molecular chaperone to help folding.

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