Inclusion bodies as reversible protein reservoirs

Abstract

Inclusion bodies (IBs) are major protein aggregates that arise owing to a deficiency of the cell's protein folding machinery. This often occurs in recombinant bacteria where plasmid-encoded genes are being highly expressed. In this context, research into the prevention of IB formation or the re-forming of correctly folded protein from IBs is of great importance to the biotechnology industry. Understanding IBs is equally important for research into amyloid diseases (such as Alzheimer's disease), which are caused by misfolding of proteins, resulting in their deposition in the cells as fibrils. Surprisingly, given the significance of IBs, they have generally been poorly investigated. However, a recent paper by Carrio et al. 1xCarrio, M.M et al. : 7–11See all References1 is an exception. They have shown that IB production in vivo is, in fact, caused by an imbalance between protein aggregation and solubility.The authors used two plasmid-encoded proteins expressed in Escherichia coli to study IB production. One was a β-galactosidase fused to a viral capsid protein, and the other was phage 22 tail-spike protein (TSP). In both systems, they calculated the number of IBs formed and their volume, the amount of soluble versus insoluble protein, and the activity of the protein as an indicator of proper folding. These measurements were taken at hourly intervals before and after the addition of chloramphenicol, which arrests protein synthesis. In the β-galactosidase system, using SDS-PAGE and densitometry against known standards, they showed that the amount of insoluble material in the cell cultures sharply decreased upon addition of chloramphenicol, with a concomitant increase in the amount of soluble material. A combination of microscopy and digital imaging also showed a reduction in the number and volume of IBs in the cells. Correct re-folding of the protein was confirmed by an increase in the β-galactosidase activity in the cultures following termination of protein synthesis. The same trend was found with the expression of TSP. Carrio et al. also demonstrated in vitro re-folding of IB proteins by incubating purified IBs from the two strains with cell extracts from plasmid-free BL26 cells, with the result being a recovery of enzymatic activity and soluble peptide in both systems.The authors concluded that IBs are the result of an imbalance in the equilibrium between protein precipitation and re-folding and solubility in the cell. By switching off protein production, the protein folding machinery can catch up, thus redressing the balance. The authors therefore see IBs not as a dead-end product caused by inefficiency of the bacterial protein folding machinery, but rather as a reversible reservoir of protein awaiting correct folding when conditions permit. The significance of the result is already apparent: IB solubility in vitro could provide the answer to the problem of IB formation in biotechnology and, from a clinical viewpoint, it raises possibilities for new therapeutic approaches to amyloid diseases.