Allen, S. C. H., Byron, A., Lord, J. M., Davey, J., Roberts, L. M. and Ladds, G. (2005) Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non-lethal ricin A chain variants. Yeast. 22(16), pp. 1287-1297. 0749-503X.
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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
Uncontrolled Keywords:
gap repair, yeast, mutation, ricin A chain
Subjects:
Creators:
Allen, S. C. H., Byron, A., Lord, J. M., Davey, J., Roberts, L. M. and Ladds, G.
Publisher:
John Wiley & Sons
Faculties, Divisions and Institutes:
Date:
December 2005
Date Type:
Publication
Page Range:
pp. 1287-1297
Journal or Publication Title:
Yeast
Volume:
22
Number:
16
Language:
English
DOI:
ISSN:
0749-503X
Status:
Published / Disseminated
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