A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

Pomerantsev, Andrei P.; Pomerantseva, Olga M.; Moayeri, Mahtab; Fattah, Rasem J.; Tallant, C.; Leppla, Stephen H.

doi:10.1016/j.pep.2011.05.016

Cited by 44 publications

(66 citation statements)

References 36 publications

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“…In addition to karilysin, only MmpZ from Bacillus anthracis has been functionally assessed at the genetic level through knockout studies in B. anthracis cells, but it has not been isolated or characterized (34). Similarly to vertebrate MMPs, karilysin showed preference for medium-sized to bulky hydrophobic residues (leucine, tyrosine and methionine) in the specificity pocket, S 1 Ј (Ref.…”

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confidence: 99%

A Novel Mechanism of Latency in Matrix Metalloproteinases

López-Pelegrín

Książek

Karim

et al. 2015

Journal of Biological Chemistry

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Background: Animal and plant matrix metalloproteinases (MMPs) are kept zymogenic through large prodomains and a cysteine-switch mechanism. Results: Bacterial MMP karilysin has only a short N-terminal peptide upstream of the catalytic domain, which lacks cysteines. Conclusion: This peptide inhibits through an aspartate-switch mechanism and also exerts other functions of authentic prodomains. Significance: Karilysin is kept latent by a novel mechanism for MMPs.

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confidence: 99%

A Novel Mechanism of Latency in Matrix Metalloproteinases

López-Pelegrín

Książek

Karim

et al. 2015

Journal of Biological Chemistry

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“…Dynabeads TALON was obtained from Invitrogen. PA and FP59 were prepared in our laboratory as described (25)(26)(27). The extracellular domain (ECD) of CMG2 (amino acids 40 -218) was produced in Escherichia coli with an N-terminal His 6 tag (17, 23).…”

Section: Methodsmentioning

confidence: 99%

“…Expression and Purification of PA Proteins-Plasmids encoding individual constructs described above were transformed into the nonvirulent B. anthracis strain BH450 or BH460, and transformants were grown in FA medium with 10 g/ml kanamycin for 15 h at 37°C (25). PA proteins were concentrated from culture supernatants and purified by chromatography on a MonoQ column (GE Healthcare) by methods described previously (25,28).…”

Section: Methodsmentioning

confidence: 99%

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Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Chen

Liu

Leysath

et al. 2016

Journal of Biological Chemistry

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The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates the translocation of the two enzymatic moieties of the toxin to the cytosol. Two PA receptors are known, with capillary morphogenesis protein 2 (CMG2) being the more important for pathogenesis and tumor endothelial marker 8 (TEM8) playing a minor role. The C-terminal PA domain 4 (PAD4) has extensive interactions with the receptors and is required for binding. Our previous study identified PAD4 variants having enhanced TEM8 binding specificity. To obtain PA variants that selectively bind to CMG2, here we performed phage display selections using magnetic beads having bound CMG2. We found that PA residue isoleucine 656 plays a critical role in PA binding to TEM8 but has a much lesser effect on PA binding to CMG2. We further characterized the role of residue 656 in distinguishing PA binding to CMG2 versus TEM8 by substituting it with the other 19 amino acids. Of the resulting variants, PA I656Q and PA I656V had significantly reduced activity on TEM8-expressing CHO cells but maintained their activity on CMG2-expressing CHO cells. The preference of these PA mutants for CMG2 over TEM8 was further demonstrated using mouse embryonic fibroblast cells and mice deficient in the CMG2 and/or the TEM8 receptors. The structural basis of the alterations in the receptor binding activities of these mutants is also discussed.Anthrax toxin is composed of three nontoxic proteins: protective antigen (PA), 3 lethal factor (LF), and edema factor (EF), which are released from Bacillus anthracis as monomers and assemble into toxic complexes on the surface of animal cells (1-3). PA is the moiety which binds to cell surface receptors and facilitates entry of the enzymatic EF and LF moieties into the host cell cytosol. Two anthrax toxin receptors have been identified; capillary morphogenesis protein 2 (CMG2, or anthrax toxin receptor 2) is the physiologically relevant toxin receptor mediating most of the in vivo toxicity of the toxin, whereas tumor endothelial marker 8 (TEM8, or anthrax toxin receptor 1) functions only as a minor anthrax toxin receptor (4 -8). Upon binding to the toxin receptors, PA is cleaved at the sequence 164 RKKR 167 by cell surface furin or furin-like proteases (9, 10), yielding the C-terminal 63-kDa fragment (PA63), which assembles to form a ring-shaped oligomer. The PA63 oligomer then binds LF and/or EF, and the toxin complex is internalized by receptor-mediated endocytosis. LF and EF are translocated into the cytosol from early or late endosomes to exert their cytotoxic effects. Therefore, PA plays the crucial role in anthrax toxin pathogenesis, serving as the delivery vehicle for translocation of LF and EF into the cytosol of the cell.The absolute requirement for cell surface proteolytic activation of PA for the toxin's action prompted our group and others to reengineer PA to make it depend on tumor-associated proteases for activation, thereby achieving high specificity for tumors (11)(12)(13)(14)(15). In a recent example, ...

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“…B. anthracis was also used as effective vector for production of recombinant proteins after deletion of six proteases (Pomerantsev et al, 2011). Experimental upshot makes it understand that protease enzyme of Bacillus anthracis carries an important active site responsible for its lethal effect.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Bacillus anthracis proteases through protein-protein interaction: an in silico study of anthrax pathogenicity

Banerjee¹,

Pal²,

Paul³

et al. 2014

TANG [HUMANITAS MEDICINE]

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Anthrax is the deadly disease for human being caused by Bacillus anthracis. Instantaneous research work on the mode of infection of the organism revealed that different proteases are involved in different steps of pathogenesis. Present study reports the in silico characterization and the detection of pathogenic proteases involved in anthrax infection through protein-protein interaction. A total of 13 acid, 9 neutral, and 1 alkaline protease of Bacillus anthracis were selected for analysing the physicochemical parameter, the protein superfamily and family search, multiple sequence alignment, phylogenetic tree construction, protein-protein interactions and motif finding. Among the 13 acid proteases, 10 were found as extracellular enzymes that interact with immune inhibitor A (InhA) and help the organism to cross the blood brain barrier during the process of infection. Multiple sequence alignment of above acid proteases revealed the position 368, 489, and 498-contained 100% conserved amino acids which could be used to deactivate the protease. Among the groups analyzed, only acid protease were found to interact with InhA, which indicated that metalloproteases of acid protease group have the capability to develop pathogenesis during B. anthracis infection. Deactivation of conserved amino acid position of germination protease can stop the sporulation and germination of B anthracis cell. The detailed interaction study of neutral and alkaline proteases could also be helpful to design the interaction network for the better understanding of anthrax disease.

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A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

Cited by 44 publications

References 36 publications

A Novel Mechanism of Latency in Matrix Metalloproteinases

A Novel Mechanism of Latency in Matrix Metalloproteinases

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Characterization of Bacillus anthracis proteases through protein-protein interaction: an in silico study of anthrax pathogenicity

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