Protein refolding using chemical refolding additives

Abstract

In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science and engineering. Inclusion-body-based protein production is a promising method because high yields are achieved in the upstream process, although the refolding of solubilized, unfolded proteins in downstream processes often leads to significantly lower yields. The most challenging problem is that the effective condition for refolding is protein dependent and is therefore difficult to select in a rational manner. Accordingly, considerable time and expense using trial-and-error approaches are often needed to increase the final protein yield. Furthermore, for certain target proteins, finding suitable conditions to achieve an adequate yield cannot be obtained by existing methods. Therefore, to convert such a troublesome refolding process into a routine one, a wide array of methods based on novel technologies and materials have been developed. These methods select refolding conditions where productive refolding dominates over unproductive aggregation in competitive refolding reactions. This review focuses on synthetic refolding additives and describes the concepts underlying the development of reported chemical additives or chemical-additive-based methods that contribute to the emergence of a universal refolding method.

Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

“…This system made it very easy to perform on-column refolding, with a continuous, gradual reduction in urea concentration, while the protein was still bound to the Ni Sepharose resin. The immobilization of a protein on a column prevents protein aggregation caused by intermolecular interactions [24–27]. To remove remaining protein impurities (Fig 2E Lane 2), the eluate from the Ni Sepharose column was subjected to a second purification step, Superdex75 gel filtration (Fig 2E Lane 3).…”

Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

“…Thus, many additives have been developed and used to improve the recovery yields of the active proteins [23]. In many cases, the inclusion body proteins have been successfully refolded.…”

Synthetic approaches to protein phosphorylation

“…The gyrase intein can function in the presence of moderate concentrations of denaturant, partly alleviating this problem, but recovering functional protein after removing denaturing agents is often challenging. [52] Despite these limitations, expressed protein ligation is a relatively attractive method for the study of protein phosphorylation as evidenced by the many cases where it has been successfully employed.…”