Protein Solubility Optimization

The expressed proteins may appear and accumulate in the form of insoluble inclusion bodies, which is a common problem in the bacterial protein expression system. In order to ensure the ultimate product quality, Creative BioMart provides a variety of solutions according to different expression conditions. customers can contact us to obtain a suitable solution as an optional service for your E.coli exogenous protein expression service.

Solution 1: Reduce the expression level appropriately.

For those high-level protein expression programs, our strategy is to:

A. Adjust the expression condition. Lower the bacteria growth temperature to restrict the protein synthesis rate.

B. Change the promoter to a weaker one.

C. Use plasmid with lower copy in host bacteria.

B. Adjust the dose and concentration of the inducer.

Solution 2: Changing the growth medium.

A. Addition of prosthetic groups or co-factors which are essential for proper folding or for protein stability.

B. Addition of buffer to control pH fluctuation in the medium during growth.

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:

Solution 3: Using specific host strains

A. AD494, which has a mutation in thioredoxin reductase (trxB).

B. Origami, a double mutant in thioredoxin reductase (trxB) and glutathione reductase.

Solution 4: In vitro denaturation and refolding of the protein

When despite all efforts the target protein still is expressed in inclusion bodies, the last resort is to denature and refold the protein in vitro. This procedure is carried out in three phases:

A. Isolation of the inclusion bodies.

B. Solubilization and denaturation of the target protein. This is done by the addition of a denaturing agent (usually guanidine or urea) under reducing conditions (e.g. 20 mM DTT).

C. Refolding of the protein by removing the denaturating agent using dialysis, dilution or chromatography. For proteins containing disulfide bonds, this has to be carried out in the presence of a redox shuttling system e.g. reduced and oxidized glutathione.

Solution 5: 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.

Solution 6: Expression of a fragment of the protein

E. coli does not express well in very large proteins (> 70 kDa). Choosing 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.

Creative BioMart is always devoted to providing high-quality and satisfactory service to our customers. If you are interested in our services or have any questions please feel free to contact us or make an online inquiry.