Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli. Infect Immun 1987 Jul;55(7):1647-51
Date
07/01/1987Pubmed ID
3110068Pubmed Central ID
PMC260572DOI
10.1128/iai.55.7.1647-1651.1987Scopus ID
2-s2.0-0023238618 (requires institutional sign-in at Scopus site) 47 CitationsAbstract
A mutant, full-length form of diphtheria toxin was cloned into Escherichia coli K-12 and expressed under BL-1 + EK-1 containment. A gene fragment encoding the catalytic domain of the toxin was subjected to oligonucleotide-directed mutagenesis to produce a three-base mutation of an active site residue; Glu-148 was thereby replaced by Ser. Ser-148 fragment A had less than 1% of the ADP-ribosyltransferase activity of wild-type fragment A. Next, the complementary portion of the toxin structural gene was spliced with the mutated DNA fragment downstream of codon 148 to produce a construct that encoded mutant whole toxin with Ser at position 148. The mutant toxin was indistinguishable from authentic diphtheria toxin by Western blot analysis, but was about 800-fold less cytotoxic than wild-type toxin for BS-C-1 cells. Evidence from subunit exchange experiments indicated that a substantial fraction of the mutant toxin contained a fully functional B moiety, capable of mediating the entry of wild-type fragment A into sensitive mammalian cells. This combination of approaches provides a means of applying recombinant DNA methods in E. coli to study structure-function relationships in whole diphtheria toxin.
Author List
Barbieri JT, Collier RJAuthor
Joseph T. Barbieri PhD Professor in the Microbiology and Immunology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
ADP Ribose TransferasesAmino Acid Sequence
Cells, Cultured
Cloning, Molecular
Diphtheria Toxin
Escherichia coli
Immunologic Techniques
Mutation
Pentosyltransferases
Protein Biosynthesis
Recombinant Proteins