A novel strategy for the purification of recombinantly expressed unstructured protein domains.

We recently found that the larger parts of the endocytic proteins epsin 1 and AP180 consist of an unstructured polypeptide chain. As a result these segments are completely heat-stable without loss of their functional properties. We have taken advantage of this fact and developed a combined heat lysis and pre-purification procedure after expressing the disordered domains in E. coli. This results in the irreversible denaturation and precipitation of the majority of bacterial proteins. The bacteria are resuspended in a non-denaturing buffer, heated in a boiling water bath and shock-cooled. We demonstrate that this procedure compared to conventional lysis improves both yield and quality of the purified protein.