In situ proteolytic digestion of inclusion body polypeptides occurs as a cascade process.
Misfolded proteins undergo a preferent degradation ruled by the housekeeping bacterial proteolytic system, but upon precipitation as inclusion bodies their stability dramatically increases. The susceptibility of aggregated polypeptides to proteolytic attack remains essentially unexplored in bacteria and also in eukaryotic cells. We have studied here the in vitro proteolysis of beta-galactosidase fusion proteins by trypsin treatment of purified inclusion bodies. A cascade digestion process similar to that occurring in vivo 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 probably on solvent-exposed surfaces of inclusion bodies. In addition, the sequence of the proteolytic attack is influenced by protein determinants other than amino acid sequence, the early digestion steps having a dramatic influence on the further cleavage susceptibility of the intermediate degradation fragments. These observations indicate unexpected conformational changes of inclusion body proteins during their site-limited digestion, that could promote protein release from aggregates, thus partially accounting for the plasticity of in vivo protein precipitation and solubilization in bacteria.
Cubarsí
R
R
Institut de Biologia Fonamental, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 08193, Spain.
Carrió
M M
MM
Villaverde
A
A
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002213:Capsid / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins; D002458:Cell Fractionation; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism*; D008954:Models, Biological; D010455:Peptides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014357:Trypsin / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D036022:Capsid Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D010450:Endopeptidases; D014357:Trypsin