During expression of many recombinant proteins, off-pathway association of partially folded intermediates into inclusion bodies competes with productive folding. A common assumption is that such aggregation reactions are nonspecific processes. The multimeric intermediates along the aggregation pathway have been identified for both the P22 tailspike and P22 coat protein. We show that for a mixture of proteins refolding in vitro, folding intermediates do not coaggregate with each other but only with themselves. This indicates that aggregation occurs by specific interaction of certain conformations of folding intermediates rather than by nonspecific coaggregation, providing a rationale for recovering relatively pure protein from the inclusion body state.

An unexpected aspect of the expression of cloned genes is the frequent failure of newly synthesized polypeptide chains to reach their native state, accumulating instead as insoluble inclusion bodies. Amyloid deposits represent a related state associated with a variety of human diseases. The critical folding intermediates at the juncture of productive folding and the off-pathway aggregation reaction have been identified for the phage P22 tailspike and coat proteins. Though the parallel beta coil tailspike is thermostable, an early intracellular folding intermediate is thermolabile. As the temperature of intracellular folding is increased, this species partitions to inclusion bodies, a kinetic trap within the cell. The earliest intermediates along the in vitro aggregation pathway, sequential multimers of the thermolabile folding intermediates, have been directly identified by native gel electrophoresis. Temperature-sensitive folding (tsf) mutations identify sites in the beta coil domain, which direct the junctional intermediate down the productive pathway. Global suppressors of tsf mutants inhibit the pathway to inclusion bodies, rescuing the mutant chains. These mutants identify sites important for avoiding aggregation. Coat folding intermediates also partition to inclusion bodies as temperature is increased. Coat tsf mutants are suppressed by overexpression of the GroE chaperonin, indicating that the thermolabile intermediate is a physiological substrate for GroE. We suggest that many proteins are likely to have thermolabile intermediates in their intracellular folding pathways, which will be precursors to inclusion body formation at elevated temperatures and therefore substrates for heat shock chaperonins.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (Speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, DjavadiOhaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.

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