Tandem folding methods to improve protein folding yield

摘要

Methods of refolding proteins are provided, especially cysteine-containing proteins such as insulin, proinsulin, and analogues thereof. The methods make used of tandem folding via the addition, at two different time, of two different reducing agents. A first reversible reducing agent can be added to induce folding and, at a later time, a second irreversible reducing agent can be added to prevent and/or reverse the formation of aggregates via intermolecular sulfide bonds. The methods can be used to reform a variety of cysteine-containing proteins with high yield of the native form, e.g. about 60 mol %, 70 mol %, or more.