1. Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non-lethal ricin A chain variants.
- Author
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Allen SC, Byron A, Lord JM, Davey J, Roberts LM, and Ladds G
- Subjects
- DNA, Fungal chemistry, DNA, Fungal genetics, Mutagenesis, Open Reading Frames genetics, Polymerase Chain Reaction, Ricin isolation & purification, Saccharomyces cerevisiae chemistry, Selection, Genetic, Transformation, Genetic, Ricin genetics, Ricin metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
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., (Copyright 2005 John Wiley & Sons, Ltd.)
- Published
- 2005
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