Polypeptide Research Renaturation of inclusion body expressed proteins

Polypeptide Research Renaturation of inclusion body expressed proteins

  1. Inclusion body:

  2. Formation of inclusion body:

  3. Favorable factors for inclusion body expression:

  4. Bacteria breaking:

  5. Separation:

  6. wash:

  7. dissolution:

Core tips:

Key words: inclusion body

Key words: inclusion body refolding protein inclusion body expression

Protein renaturation is a worldwide problem. There is no general method or even reliable law, so we can only try.

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Refolding of inclusion body expressed proteins

Inclusion body:

Inclusion body refers to the aggregation of proteins expressed by bacteria in cells to form inactive solid particles.

Composition and characteristics of inclusion body:

Generally, it contains more than 50% recombinant proteins, the rest are ribosome elements, RNA polymerases, outer membrane proteins OmpC, OmpF and OmpA, etc., circular or notched plasmid DNA, as well as liposomes, lipopolysaccharides, etc., which are 0.5-1um in size, insoluble and water-soluble, only soluble in denaturants such as urea, guanidine hydrochloride, etc.

Formation of inclusion body:

It is mainly due to the lack of some protein folding cofactors in the expression process of recombinant protein, or the environment is not suitable to form the correct secondary bond.

  1. It was found that inclusion bodies were seldom formed when the expression was too high, and the higher the expression was, the easier inclusion bodies were formed. The reason may be that the synthesis speed is too fast, so there is not enough time for folding, disulfide bond can not be correctly matched, too many non-specific binding between proteins, protein can not achieve enough solubility, etc.
  2. Amino acid composition of recombinant protein: Generally speaking, the more sulfur-containing amino acids are, the easier inclusion bodies are to be formed, and the content of proline is positively correlated with the formation of inclusion bodies.
  3. The environment of recombinant protein: inclusion bodies are easy to form when the fermentation temperature is high or the intracellular pH is close to the isoelectric point of the protein.
  4. Recombinant proteins are heterologous proteins of E.coli. Due to the lack of post-translational modification enzymes in eukaryotes, a large number of intermediates are accumulated, which is easy to form inclusion body precipitation. Therefore, co expression of molecular chaperones has been used to increase the proportion of soluble proteins.

Favorable factors for inclusion body expression:

  1. The expression of inclusion body can avoid the degradation of exogenous protein by protease.
  2. The concentration of extracellular protein was decreased, which was beneficial to the increase of expression.
  3. The content of heteroprotein in inclusion body is low, and it can be separated from soluble protein by simple low-speed centrifugation, which is good for separation and purification.
  4. It is not sensitive to mechanical agitation and ultrasonic breaking, and is easy to break the wall and separate from cell membrane fragments.

Bacteria breaking:

  1. Mechanical crushing
  2. Ultrasonic breaking
  3. Chemical crushing

Separation:

  1. Centrifugation: centrifugation of 5000-20000 g for 15 min can precipitate most inclusion bodies and separate them from soluble proteins.
  2. Filtration or extraction method:

wash:

Due to the adhesion of liposome, some broken cell membrane and membrane protein with inclusion body, inclusion body should be washed before dissolving inclusion body, usually with low concentration denaturant such as 2m urea in 50mm trisph7.0-8.5, 1mmedta. In addition, Triton X-100, a mild detergent, can be used to remove membrane fragments and protein.

dissolution:

The common denaturants are urea (8m) and guanidine hydrochloride (gdnhcl6-8m). Through the interaction between ions, the hydrogen bond between inclusion body proteins is destroyed and the proteins are solubilized. Among them, the solubilization effect of urea is less poor, and guanidine isocyanate (gdnscn) is the strongest.

Detergents: such as strong anionic detergents SDS, can destroy the water bonds in proteins, and can solubilize almost all proteins. The problem is that SDS cannot be completely removed and is not allowed to be used in the pharmaceutical process.

Extreme pH: it can destroy the secondary bond of protein and solubilize protein. If someone dissolves bovine growth hormone and bovine chymotrypsin inclusion body at pH > 9.0. Some proteins can be dissolved in 60mmhcl. These methods are only suitable for solubilization of a few proteins.

Use concentration and action time of denaturant: generally in the alkaline environment such as ph8.0-9.0, urea is not stable in the alkaline environment, generally not more than ph1.0. Some proteins can only be used with guanidine hydrochloride such as IL-4. When solubilizing, it is usually overnight at room temperature, but guanidine hydrochloride can completely denature and dissolve most proteins at 37 ℃ for 1 hour.

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