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Department of Pharmacology

Allen, SCH, Byron, A, Lord, JM, Davey, J, Roberts, LM, Ladds, G

Knowledge of the uptake, membrane translocation, refolding and ribosome interaction of the ribosome-inactivating toxin ricin is incomplete at the present time. Ricin A chain (RTA) is the catalytic subunit of holotoxin and is also of particular interest as a vaccine candidate. For many studies into the uptake and immunological applications of ricin, it is essential to have inactive variants. Here, following error-prone polymerase chain reaction of the RTA open reading frame, we have used a modified gap-repair protocol in Saccharomyces cerevisiae to show that it is possible to rapidly generate a panel of inactive RTA mutants. Since yeast cells have ribosomes that are highly sensitive to RTA, we utilized a genetic selection based on the viability of transformants. This enabled the recovery of a number of mutations, some not previously identified, which permitted production of full-length but non-toxic RTA proteins. Such disarmed toxins may have utility as tools to study the cytosolic entry and action of RTA, and as potential vaccine candidates.

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December 2005
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Journal articles
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