In situ proteolytic digestion of inclusion body polypeptides occurs as a cascade process

Misfolded proteins undergo a preferent degradation ruled by the housekeepin g bacterial proteolytic system, but upon precipitation as inclusion bodies their stability dramatically increases. The susceptibility of aggregated po lypeptides to proteolytic attack remains essentially unexplored in bacteria and also in eukaryotic cells. We have studied here the in vitro proteolysi s of beta -galactosidase fusion proteins by trypsin treatment of purified i nclusion bodies. A cascade digestion process similar to that occurring in v ivo has been observed in the insoluble fraction of the digestion reaction. This suggests that major protease target sites are not either lost or newly generated by protein precipitation and that the digestion occurs in situ p robably on solvent-exposed surfaces of inclusion bodies. In addition, the s equence of the proteolytic attack is influenced by protein determinants oth er than amino acid sequence, the early digestion steps having a dramatic in fluence on the further cleavage susceptibility of the intermediate degradat ion fragments. These observations indicate unexpected conformational change s of inclusion body proteins during their site-limited digestion, that coul d promote protein release from aggregates, thus partially accounting for th e plasticity of in vivo protein precipitation and solubilization in bacteri a, (C) 2001 Academic Press.