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 a-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 Ion 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°C but could not be released by the same procedures after growth at 42°C. On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30°C by isopropyl-(B-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.Diphtheria toxin (for a review, see reference 42) is a 535-amino-acid polypeptide (27,32,45) that is secreted in amounts as large as 500 mg/liter by toxinogenic strains of Corynebacterium diphtheriae grown under appropriate conditions (46). The toxin can be resolved into two fragments by mild proteolysis and disulfide reduction (17, 21). Fragment A (Fig.

Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S layer of Thermus thermophilus HB8

“…Low-level expression of pMF51 in the absence of IPTG was most likely due to transcriptional leakiness, as indicated by the inability of pMF51 to express P1OO-derived proteins in 38 -FIG. 4. Expression of pMF51 in E. coli BL21 DE3.…”

Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.

“…IL-2-toxin expressed and secreted into the culture supernatant fluid of yeast dph mutants has an apparent M, of 68,000 and was Detailed studies of the properties of chimeric toxins expressed in E. coli have been impeded by low levels of synthesis, insolubility, and proteolytic degradation (16). Only by employing protease-deficient strains and a strong inducible promoter, conducting growth at low temperatures, and directing the expression of the chimeric toxin to the cytoplasm has it been possible to achieve efficient expression and recovery of intact proteins in E. coli (32).…”

Expression and Immunogenicity of a Recombinant Diphtheria Toxin Fragment A in Streptococcus gordonii

“…The correct fusions of SpaP-DTA, SpaP-DTA 2 , and SpaP-DTA 3 carried on pDTA, pDTA 2 , and pDTA 3 were verified by restriction analysis (data not shown). E. coli cells harboring these plasmids were shown to express the expected 207-, 229-, and 251-kDa fusion protein, respectively, that were recognized by the mouse anti-DT and rabbit anti-P1 antibodies (data not shown).…”

“…Expression of full-length DT and fragments of DT has been described in Escherichia coli (1,2,3), Salmonella enterica (7,29), Staphylococcus carnosus (6), and Mycobacterium bovis (25). In E. coli and S. enterica, the recombinant protein was immunogenic but the antisera contained only weak neutralizing activity.…”