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[3] Isolating inclusion bodies from bacteria

1999; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(99)09005-9

1557-7988

George Georgiou, Pascal Valax,

Protein Structure and Dynamics

In 1975 Prouty et al. first described the formation of dense, amorphous intracellular granules in Escherichia coli cells grown in the presence of the amino acid analog canavanine. These granules, comprising primarily of polypeptide chains, could be solubilized by sodium dodecyl sulfate (SDS) and were not surrounded by any sort of membrane. For several years this observation was considered an aberrant, and rather irrelevant, cellular response induced by growth under nonphysiological conditions. It was not until much later that it became apparent that protein aggregation in vivo is a widespread phenomenon, manifested in cells overexpressing heterologous proteins or native proteins beyond a certain level, and in cells exposed to thermal or other kinds of physiological stress. In addition, mutations resulting in amino acid substitutions, deletions, or insertions can interfere with the folding of a polypeptide to the native state, causing the formation of protein aggregates. Intracellular protein aggregates form dense, electron-refracting particles that can be distinguished readily from other cell components by electron microscopy. For this reason, protein aggregates, at least those observed in microorganisms, are usually called inclusion bodies or, less often, refractile bodies. However, protein misfolding and self-association may occur even when inclusion bodies cannot be detected by electron microscopy or by following careful cell fractionation. Nonetheless, for practical purposes, it is safe to assume that the expression of proteins susceptible to aggregation at a level 2% or greater of the total cell protein will be accompanied by the appearance of readily identifiable inclusion bodies.