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.
%0 Journal Article
%1 YeastRicin
%A Allen, Stuart C. H.
%A Byron, Adam
%A Lord, J. Michael
%A Davey, John
%A Roberts, Lynne M.
%A Ladds, Graham
%D 2005
%J Yeast
%K A chain gap mutation repair ricin yeast
%N 16
%P 1287-1297
%T Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non-lethal ricin A chain variants.
%U http://dx.doi.org/10.1002/yea.1330
%V 22
%X 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.
@article{YeastRicin,
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.},
added-at = {2009-08-21T10:07:53.000+0200},
author = {Allen, Stuart C. H. and Byron, Adam and Lord, J. Michael and Davey, John and Roberts, Lynne M. and Ladds, Graham},
biburl = {https://www.bibsonomy.org/bibtex/20a85243bf8c26a9a8642eeaa3ea22c8c/adambyron},
interhash = {d683701f0630009778cedf7201a06bb5},
intrahash = {0a85243bf8c26a9a8642eeaa3ea22c8c},
journal = {Yeast},
keywords = {A chain gap mutation repair ricin yeast},
month = {December},
number = 16,
pages = {1287-1297},
timestamp = {2009-08-21T10:22:35.000+0200},
title = {Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non-lethal ricin A chain variants.},
url = {http://dx.doi.org/10.1002/yea.1330},
volume = 22,
year = 2005
}