Choline‐binding domain as a novel affinity tag for purification of fusion proteins produced in <i>Pichia pastoris</i>

Caubín, J.; Martı́n, Humberto; Roa, Alejandra A; Cosano, Inmaculada C.; Pozuelo, Mercedes; Fuente, J. M. de la; Sánchez-Puelles, José María; Molina, M.; Nombela, César

doi:10.1002/bit.1106

Cited by 16 publications

(13 citation statements)

References 19 publications

(23 reference statements)

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“…To assess whether lysin SMl demonstrated its predicted biological activities, we first examined its binding to DEAE-cellulose, a property that is a hallmark of choline-binding proteins [21]. Lysin SM1 , N-lysin SM1 , and C-lysin SM1 were expressed individually in Escherichia coli , and lysates from these strains were applied to a DEAE-cellulose column [16].…”

Section: Resultsmentioning

confidence: 99%

“…The bacteria were incubated with purified FLAG lysin SM1 (0 to 10 µg/ml) for 30 min at room temperature. The samples were washed twice with PBS to remove unbound FLAG lysin SM1 and incubated with PBS-2% choline chloride to elute choline-binding proteins from the cell walls, as described previously [21]. Eluted cell wall proteins were harvested by centrifugation and loaded onto SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

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Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

et al. 2010

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The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

et al. 2010

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“…Both PblA and PblB contain repeat regions of glycine‐tryptophan‐rich motifs, similar to the choline binding proteins of S. pneumoniae , and the lipoteichoic acid binding regions of InlB of Listeria monocytogenes (Jonquieres et al ., 1999). Moreover, we have shown that PblA and PblB can be purified from culture media by affinity chromatography with DEAE cellulose (a choline analogue), as has been done with other choline binding proteins (Sanchez‐Beato et al ., 1995; Caubin et al ., 2001; Fan et al ., 2004; Moldes et al ., 2004).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB

Mitchell

Siboo

Takamatsu

et al. 2007

Molecular Microbiology

111

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SummaryPblA and PblB are prophage-encoded proteins of Streptococcus mitis strain SF100 that mediate binding to human platelets. The mechanism for surface expression of these proteins has been unknown, as they do not contain signal sequences or cell wall sorting motifs. We therefore assessed whether expression of these proteins was linked the lytic cycle of the prophage. Deletion of either the holin or lysin gene resulted in retention of PblA and PblB in the cytoplasm, and loss of these proteins from the cell wall. Flow cytometric analysis revealed that induction of phage replication in SF100 produced a subpopulation of cells with increased permeability. This effect was abrogated by disruption of the holin and lysin genes. Treatment of these mutants with exogenous PblA and PblB restored surface expression, apparently via binding of the proteins to cell wall choline. Loss of PblA and PblB expression was associated with decreased platelet binding in vitro, and reduced virulence in an animal model of endocarditis. Thus, expression of PblA and PblB occurs via a novel mechanism, whereby phage induction increases bacterial permeability and release of the proteins, followed by their binding to surface of viable cells. This mechanism may be important for endovascular infection.

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“…To get this, we made use of the affinity tag CLytA, which is the choline-binding module of the amidase N-acetylmuramoyl-L-alanine (LytA) from Streptococcus pneumoniae [15]. The CLytA domain shows high affinity for choline and choline structural analogs, such as diethylaminoethanol (DEAE), and it is routinely used as an affinity tag for the single-step purification and immobilization of fusion proteins [16][17][18].The chimera was specifically immobilized onto MNPs functionalized with DEAE. Both, free and immobilized, CLytA-DAAO chimera are able to induce cell death by increasing ROS production, which caused DNA damage in several colon carcinoma and pancreatic adenocarcinoma cell lines as well as in glioblastoma cell lines derived from primary cultures obtained in our laboratory directly from glioblastoma patients, at doses that are safe for non-tumor cells.…”

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confidence: 99%

CLytA-DAAO, Free and Immobilized in Magnetic Nanoparticles, Induces Cell Death in Human Cancer Cells

et al. 2020

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D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair.

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Choline‐binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris

Cited by 16 publications

References 19 publications

Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB

CLytA-DAAO, Free and Immobilized in Magnetic Nanoparticles, Induces Cell Death in Human Cancer Cells

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