Expression and purification of cysteine introduced recombinant saporin

Günhan, Emine; Swe, Mimi; Palazoglu, Mine; Voss, John C.; Chalupa, Leo M.

doi:10.1016/j.pep.2007.11.005

Cited by 7 publications

(9 citation statements)

References 28 publications

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“…This last step is crucial because an inefficient elimination of imidazole employed for the protein elution significantly compromises the freeze–thaw stability of C-Sap. The purification protocol used here was designed to allow the obtainment of around 2.8 mg of C-Sap/L culture, similar to what was previously reported [ 26 ].…”

Section: Resultsmentioning

confidence: 99%

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells

et al. 2022

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Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin’s main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.

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Section: Resultsmentioning

confidence: 99%

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells

et al. 2022

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“…Commercial saporin is isolated directly from seeds of the Saponaria officinalis plant, whereas our in-house product is a recombinant version. 27 Our laboratory has optimized the production and purification of recombinant saporin, as well as the bioconjugation of saporin to streptavidin for immunotoxin generation. Figure S1 B shows a representative Coomassie-stained polyacrylamide gel of biotinylated CD117 mAb, commercial saporin, in-house-generated recombinant saporin, and their associated CD117-sap products after conjugation.…”

Section: Resultsmentioning

confidence: 99%

“…Biotinylated anti-CD117 (clone 2B8, Biolegend, San Diego, CA, USA) was combined at a 1:1 molar ratio with either streptavidin-ZAP (ATS, San Diego, CA, USA) or an in-house-developed streptavidin-saporin product. 27 CD117-saporin immunotoxin was delivered by retro-orbital injection at a dose of 0.5 mg/kg on day −5 before HSCT. Rabbit anti-mouse ATG (Cedarlane, Burlington, NC, USA) was administered by intraperitoneal injection at a dose of 30 mg/kg on day −5.…”

Section: Methodsmentioning

confidence: 99%

Non-genotoxic conditioning facilitates hematopoietic stem cell gene therapy for hemophilia A using bioengineered factor VIII

Russell

Prince

Lundgren

et al. 2021

Molecular Therapy - Methods & Clinical Development

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Hematopoietic stem and progenitor cell (HSPC) lentiviral gene therapy is a promising strategy toward a lifelong cure for hemophilia A (HA). The primary risks associated with this approach center on the requirement for pre-transplantation conditioning necessary to make space for, and provide immune suppression against, stem cells and blood coagulation factor VIII, respectively. Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA alkylation, that increase risk of sterility, infection, and developing secondary malignancies. In the current study, we describe a non-genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies to provide transient immune suppression against the transgene product in a murine HA gene therapy model. This strategy provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor VIII variant, termed ET3. Factor VIII procoagulant activity levels were durably elevated into the normal range and phenotypic correction achieved. Furthermore, no immunological rejection or development of anti-ET3 immunity was observed. These preclinical data support clinical translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for HA.

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“…Thus, many different toxins have been used to construct immunotoxins, e.g. ricin chain A , nigrin b , gelonin , saporin , Diphtheria toxin , Pseudomonas toxin , anthrax toxin , barnase or α‐sarcin , which behave as type I toxins. On the other hand, tumor necrosis factor and sTRAIL are type II toxins, while actinoporins , cyt A endotoxin and cecropin belong to the group of type III toxins.…”

Section: Introductionmentioning

confidence: 99%

α‐sarcin and RNase T1 based immunoconjugates: the role of intracellular trafficking in cytotoxic efficiency

et al. 2014

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Toxins have been thoroughly studied for their use as therapeutic agents in search of an improvement in toxic efficiency together with a minimization of their undesired side effects. Different studies have shown how toxins can follow different intracellular pathways which are connected with their cytotoxic action inside the cells. The work herein presented describes the different pathways followed by the ribotoxin a-sarcin and the fungal RNase T1, as toxic domains of immunoconjugates with identical binding domain, the single chain variable fragment of a monoclonal antibody raised against the glycoprotein A33. According to the results obtained both immunoconjugates enter the cells via early endosomes and, while a-sarcin can translocate directly into the cytosol to exert its deathly action, RNase T1 follows a pathway that involves lysosomes and the Golgi apparatus. These facts contribute to explaining the different cytotoxicity observed against their targeted cells, and reveal how the innate properties of the toxic domain, apart from its catalytic features, can be a key factor to be considered for immunotoxin optimization.

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Expression and purification of cysteine introduced recombinant saporin

Cited by 7 publications

References 28 publications

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells

Non-genotoxic conditioning facilitates hematopoietic stem cell gene therapy for hemophilia A using bioengineered factor VIII

α‐sarcin and RNase T1 based immunoconjugates: the role of intracellular trafficking in cytotoxic efficiency

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