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KMID : 0359319970370030541
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Korean Journal of Veterinary Research
1997 Volume.37 No. 3 p.541 ~ p.546
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Site-specific and deletional mutagenesis for two regions of Verotoxin-2 A gene encoding enzymatically active domain
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Kim Yong-Hwan
Kim Sang-Hyun
Cha In-Ho
Kim Kyoung-Shook
Lee Young-Choon
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Abstract
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There are two conserved regions with a significantly high amino acid sequence homology among the A subunits of STX, SLTs and ricin. To produce an inactive Verotoxin-2 (VT-2), two different mutants, pE167D and pDE5A, were constructed by site-directed mutagenesis, respectively, on the basis of the previous reports that two regions lie within the active-site clefts of the A subunits of ricin and STX family. The cytotoxicity (10^3CD50/ml) of VT-2 holotoxin with E167D mutation was reduced by 103-fold compared with wild-type level. In addition, VT-2 with DE5A (Trp202GlyArgIleSer206) deletion mutation showed a significantly low cytotoxicity (10^1CD50/ml), resulting in 10^5- and 10^2- fold reductions, respectively, compared with the wild-type and E167D mutatant. SDS-PAGE for protein samples showed a 33-kDa band corresponding to the A subunit of VT-2. These results indicate that reduction in cytotoxic activity was affected not by amount of VT-2 protein produced but by mutation.
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KEYWORD
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verotoxins , VTe , mutation , cytotoxicity
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