Cloned Fragment A of Diphtheria Toxin Is Expressed and Secreted into the Periplasmic Space of
            <i>Escherichia coli</i>
            K12

Leong, D; Coleman, K.; Murphy, John R.

doi:10.1126/science.6403984

Cited by 41 publications

(25 citation statements)

References 20 publications

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“…Because the three proteolytically sensitive arginines present in the Cys186-Cys20, disulfide loop of the toxin were also present in this fragment, the doublet probably arises from partial cleavage at one of these sites to yield fragment A alone. This cleavage reaction has been well demonstrated with the pDT201 protein in vitro (31).…”

Section: Resultsmentioning

confidence: 92%

“…The expression of genes for fragment A in E. coli gives a stable, active protein (31). In marked contrast, our genes coding for proteins composed of fragment A plus 97, 184, or 292 amino acids of fragment B give many intermediatelength polypeptides, in addition to the full-length species, upon expression in E. coli.…”

Section: Discussionmentioning

confidence: 99%

“…2, and their use in constructing some of the truncated toxin genes has been described previously (33a, 36). The plasmids which we modified with these linkers were constructed by Leong et al (30). pDT201 contains an 831-bp Sau3AI restriction fragment that carries the tox promoter, signal sequence, and all of the fragment A-encoding sequences.…”

Section: Resultsmentioning

confidence: 99%

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

Bishai¹,

Miyanohara²,

Murphy³

1987

J Bacteriol

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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 a-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (pto.) 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 c1857 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.

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Section: Resultsmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

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

Bishai¹,

Miyanohara²,

Murphy³

1987

J Bacteriol

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“…However, we observed the same results with sonic extracts or lysozyme-EDTA-treated cells carrying pABM1508 (PR driven, periplasmic). This observation was a surprise since we (35) and others (54) had shown that fragment A alone is expressed and secreted to the periplasmic space in a soluble form.…”

Section: Fig 3 Westem Blot Analysismentioning

confidence: 84%

High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli

Bishai¹,

Rappuoli²,

Murphy³

1987

J Bacteriol

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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.

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“…The C. diphtheriae tox 176 lysogen was grown and lysed after penicillin treatment (15), and chromosomal DNA was isolated. To detect the tox 176 sequence, restriction digests of the DNA were subjected to Southern hybridization (16) by using a probe derived from the wild-type DT-A coding sequence, subcloned from pDT201 (10). This analysis (data not shown) revealed a single hybridizing band of ca.…”

mentioning

confidence: 99%

Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

Maxwell¹,

Maxwell²,

Glode³

1987

Mol. Cell. Biol.

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DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTHl-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTHl, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15-to 30-fold more pTHl-176 than pTHl was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.

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Cloned Fragment A of Diphtheria Toxin Is Expressed and Secreted into the Periplasmic Space of Escherichia coli K12

Cited by 41 publications

References 20 publications

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

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

High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli

Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

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