Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B

William Ramses Bishai, A. Miyanohara, J. R. Murphy

Research output: Contribution to journalArticle

Abstract

We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin. These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells. Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with α-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (p(tox)) with the lambda p(R) promoter in an effort to overexpress these proteins. The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the p(R) promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated M(r) on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from p(tox) leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the p(R) promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells. It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.

Original languageEnglish (US)
Pages (from-to)1554-1563
Number of pages10
JournalJournal of Bacteriology
Volume169
Issue number4
StatePublished - 1987
Externally publishedYes

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Diphtheria Toxin
Organism Cloning
Escherichia coli
Amino Acids
Peptide Hydrolases
ADP Ribose Transferases
Melanocyte-Stimulating Hormones
Peptides
Diphtheria
Gene Fusion
Disulfides
Genes
Cysteine
Proteins
Clone Cells
Hot Temperature
Western Blotting
Ligands
Membranes
Growth

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B. / Bishai, William Ramses; Miyanohara, A.; Murphy, J. R.

In: Journal of Bacteriology, Vol. 169, No. 4, 1987, p. 1554-1563.

Research output: Contribution to journalArticle

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