Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non-lethal ricin A chain variants.
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.
Allen
Stuart C H
SC
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Byron
Adam
A
Lord
J Michael
JM
Davey
John
J
Roberts
Lynne M
LM
Ladds
Graham
G
D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D016296:Mutagenesis; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D012276:Ricin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012641:Selection, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D012276:Ricin