Korean Journal of Veterinary Research 1997;37(3):541-546.
Site-specific and deletional mutagenesis for two regions of Verotoxin-2 A gene encoding enzymatically active domain
Yong-hwan Kim1, Sang-hyun Kim2, In-ho Cha2, Kyoung-shook Kim2, Young-choon Lee2
1College of Veterinary Medicine, Gyeongsang National University
2Molecular Glycobiology Unit, Korea Research Institute of Bioscience & Biotechnology(KRIBB), KIST
Verotoxin-2 A 유전자의 효소활성 부위에 대한 위치특이적 변이 및 결손변이유발
김용환1, 김상현2, 차인호2, 김경숙2, 이영춘2
1경상대학교 수의과대학
2한국과학기술연구원 생명공학연구소 당쇄생물학 연구 Program
Abstract
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 ($10^3$ $CD_{50}/ml$) of VT-2 holotoxin with E167D mutation was reduced by $10^3$-fold compared with wild-type level. In addition, VT-2 with DE5A ($Trp_{202}GlyArgIleSer_{206}$) deletion mutation showed a significantly low cytotoxicity ($10^1$ $CD_{50}/ml$), 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.
Key Words: verotoxins, VTe, mutation, cytotoxicity
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