[20] Solubilization of protein aggregates

Publisher Summary This chapter describes the solubilization of protein aggregates. From a purification standpoint, the accumulation of protein in an aggregated form is advantageous. After breaking open the cells and centrifuging the resulting lysate, the aggregated protein can be recovered in the pellet fraction about 50% pure, although mostly in an inactive form. The majority of protein contained within these inclusion bodies is in a denatured form, in part because of the reducing environment of the E . coli cytoplasm. In addition, dimers and higher molecular weight multimers may be present. The concentration of protein in the refolding solution also affects the yield of recoverable active protein. The most significant loss during refolding of concentrated protein solutions is because of aggregate formation, which is frequently caused by covalent modifications of the unfolded protein molecules, such as intermolecular disulfide formation. The fact that many of the eukaryotic proteins expressed in E . coli are insoluble is an advantage because the isolation of inclusion bodies in itself can be a very efficient purification step. However, to a varying degree, protein contaminants do remain after inclusion body isolation by centrifugation coupled with washing procedures.