Methods for the Refolding of Disulfide-Rich Proteins.

Eschericia coli remains the workhorse producing recombinant proteins given its ease of handling, access and genetic manipulation using standard laboratory techniques. However, disulfide-rich proteins can be difficult to produce in E. coli, in large part due to the reducing environment of the bacterial cytoplasm. Refolding from insoluble inclusion bodies can be a viable strategy for generating substantial quantities of disulfide-rich protein. For the best chance of successfully refolding a protein, it is vital to carry out a variety of small-scale test refolds under a swathe of conditions including altering the concentration of urea, salts, reduced and oxidized glutathione, temperature, length of refold time and protein dilution factor. Once a protein has undergone refolding it is vital to determine that the final product is natively folded given the chance of soluble misfolded protein. For determination of correct folding, a variety of techniques can be employed, and ideally, numerous should be used together. For proteins that possess enzymatic function the gold standard to assess correct folding is an activity assay. Non-enzymatic proteins can be assessed using a combination of circular dichroism and nuclear magnetic resonance spectroscopy. These techniques should be utilized alongside mass spectrometry, Western blotting and SDS-PAGE.