Site-specific and deletional mutagenesis for two regions of Verotoxin-2 A gene encoding enzymatically active domain

Verotoxin-2 A 유전자의 효소활성 부위에 대한 위치특이적 변이 및 결손변이유발

  • Kim, Yong-hwan (College of Veterinary Medicine, Gyeongsang National University) ;
  • Kim, Sang-hyun (Molecular Glycobiology Unit, Korea Research Institute of Bioscience & Biotechnology(KRIBB), KIST) ;
  • Cha, In-ho (Molecular Glycobiology Unit, Korea Research Institute of Bioscience & Biotechnology(KRIBB), KIST) ;
  • Kim, Kyoung-shook (Molecular Glycobiology Unit, Korea Research Institute of Bioscience & Biotechnology(KRIBB), KIST) ;
  • Lee, Young-choon (Molecular Glycobiology Unit, Korea Research Institute of Bioscience & Biotechnology(KRIBB), KIST)
  • 김용환 (경상대학교 수의과대학) ;
  • 김상현 (한국과학기술연구원 생명공학연구소 당쇄생물학 연구 Program) ;
  • 차인호 (한국과학기술연구원 생명공학연구소 당쇄생물학 연구 Program) ;
  • 김경숙 (한국과학기술연구원 생명공학연구소 당쇄생물학 연구 Program) ;
  • 이영춘 (한국과학기술연구원 생명공학연구소 당쇄생물학 연구 Program)
  • Received : 1997.05.08
  • Published : 1997.09.25

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.

Acknowledgement

Supported by : Ministry of Education