In many cases the expressed protein is insoluble and accumulates in so-called inclusion bodies. This is especially true under conditions of high level expression. Several strategies are available to improve the solubility of the expressed protein.
Reducing the rate of protein synthesis. This can be done by:
Changing the growth medium.
Co-expression of chaperones and/or foldases. Two classes of proteins play an important role in in vivo protein folding.
Molecular chaperones promote the proper isomerization and cellular targeting by transiently interacting with folding intermediates. The best characterized E. coli systems are:
Foldases accelerate rate-limiting steps along the folding pathway. Three types of foldases play an important role:
Co-expression of one or more of these proteins with the target protein could lead to higher levels of soluble protein. The levels of co-expression of the different chaperones/foldases have to be optimized for each individual case. DsbA and DsbC have also shown possitive effects on expression levels when used as a fusion partner.
Periplasmic expression. Secretion of the target protein to the periplasm has a number of distinct advantages:
Secretion is achieved by the addition of a leader sequence (signal peptide) to the N-terminus of the target protein. Most used leader sequences are pelB and ompT. Unfortunately, expression yield are usually much lower and not all expressed protein is secreted into the periplasm but is also found in the medium, the cytoplasm and the cytoplasmic membrane.
Using specific host strains. The solubility of disulfide bond containing protein can be increased by using a host strain with a more oxidizing cytoplasmic environment. Two strains are commercially available (Novagen):
Addition of a fusion partner. Fusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner often improves the solubility of the fusion protein.
Expression of a fragment of the protein. E. coli does not express well very large proteins (> 70 kDa). Chosing a smaller fragment of the target protein can improve expression levels and solubility.
The solubility of a poorly soluble (or insoluble) protein can also be improved by selecting only a soluble domain for expression.
In vitro denaturation and refolding of the protein. When despite all efforts the target protein still is expressed in inclusion bodies, then the last resort is to denature and refold the protein in vitro. This procedure is carried out in three phases:
See also: In vitro denaturation and refolding.
Last updated: 12 Jan 2001