Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B.
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 alpha-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR 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 pR promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the pR 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.
D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic
D004274:DNA, Recombinant; D004167:Diphtheria Toxin; D010446:Peptide Fragments; C039171:diphtheria toxin fragment B; D010447:Peptide Hydrolases