BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Preparation of Diphtheria Toxin A Chain from Escherichia coli

  • Lee, Jong-Soo (Department of Chemistry, Kangnung National University) ;
  • Yoon, Kyoung-Bum (Department of Chemistry, Kangnung National University) ;
  • Park, Jong-Won (Department of Chemistry, Kangnung National University) ;
  • Choi, Suk-Jung (Department of Chemistry, Kangnung National University)
  • Received : 1997.01.15
  • Published : 1997.03.31
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Abstract

An expression vector was constructed containing the gene encoding diphtheria toxin A (DTA) which was placed after a T7 promoter. Cytoplasmic expression of the DTA gene resulted in the formation of an insoluble inclusion body. The inclusion body was collected after the complete lysis of the cell, and subsequent washing with 0.1% Triton X-100 released 16~30% of DTA protein from the inclusion body along with other contaminating proteins. The released DTA protein was purified by dialysis. The remaining pellet was dissolved in 8 M urea containing 5% ${\beta}-mercaptoethanol$, and the denatured DTA was renatured by the dilution-dialysis method. The total yield was 35%, and about 5 mg DTA was obtained from 1 L culture. The DTA protein has a free sulfhydryl group exposed to the protein surface, and was shown to have a tendency to dimerize through disulfide formation in the absence of ${\beta}-mercaptoethanol$. The utility of the sulfhydryl group was tested for the construction of recombinant toxins.

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