title: High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli.
abstract: ABM508 is a recombinant fusion protein consisting of the N-terminal 485 amino acids of diphtheria toxin joined to alpha-melanocyte-stimulating hormone. When expressed in Escherichia coli under the control of the tox promoter and signal sequence, ABM508 is severely degraded. When overexpressed from a thermoinducible lambda pR promoter fusion, ABM508 is largely insoluble. We compared the expression of ABM508 (501 amino acids) to a full-length mutant form of the toxin (CRM197; 535 amino acids) and found that CRM197 showed minimal proteolysis. Thus, the removal of the C-terminal 50 amino acids of the toxin destabilizes the protein, making it a target for proteases. Proteolysis of ABM508 could be reduced by removal of the tox signal sequence (thereby directing the protein to the cytoplasm) and growth in lon and htpR mutant strains of E. coli. We also showed that the solubility of tox gene products expressed in E. coli was directly related to the growth temperature of the culture. Thus, a fragment A fusion protein (223 amino acids), ABM508, and CRM197 were found in soluble extracts when expressed at 30 degrees C but could not be released by the same procedures after growth at 42 degrees C. On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30 degrees C by isopropyl-beta-D-thiogalactoside) and expressed this construct in a lon htpR strain of E. coli. This plasmid made 10 mg of soluble tox protein per liter of culture (7.7% of the total cell protein) or 14 times more than our previous maximal level. Extracts from lon htpR cells harboring this plasmid had high levels of ADP-ribosyltransferase activity, and although proteolysis still occurred, the major tox product corresponded to full-length ABM508.
authors.affiliation: Evans Department of Clinical Research, University Hospital, Boston, Massachusetts 02118.
authors.forename: W R
authors.initials: WR
authors.lastname: Bishai
authors.forename: R
authors.initials: R
authors.lastname: Rappuoli
authors.forename: J R
authors.initials: JR
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004167:Diphtheria Toxin / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005838:Genotype; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013816:Thermodynamics; D014158:Transcription, Genetic
chemical_list: D004167:Diphtheria Toxin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases