Genetic Construction of a Phosphorylation Site in Ricin A Chain: Specific Radiolabeling of Recombinant Proteins for Localization and Degradation Studies

Fryxell, Daniel K.; Li, B.Y.; Mohanraj, D.; Johnson, Betty H.; Ramakrishnan, Suresh Krishna

doi:10.1006/bbrc.1995.1654

Cited by 6 publications

(2 citation statements)

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“…We used the technique of site-specific mutation to introduce phosphorylation sites into the amino acid sequences of a number of interferons (47,48,82) (also R. Donnelly and S. Pestka, unpublished data for IL-2; W. Wu and S. Pestka, unpublished data for monoclonal antibody chCC49-WW5). The fusion approach was also useful for a variety of proteins and kinase sites (1,3-7, 9, 15,19,28,32,33,36,49-52,59,60,62,63,68,74,81,83,85,86,88): interferon (88), monoclonal antibodies (49)(50)(51)(52), retinoblastoma protein (1,32), osteopontin (4), c-Fos, (5), calmodulin (15), diphtheria toxin (59), endothelial growth factors (59), enterotoxins, (59) lymphokines (59), ricin (19,59), antibody fragments (62,63), microtubule associated protein (81), Escherichia coli RNA polymerase β′ and σ subunits (3,6), green fluorescent protein ( 85), E. coli TraT-RHO surface protein fusion (7), bacteriophage T4 primase (28), erythropoietin (68), opioid receptor peptide ligands (36), interleukin receptor subunit extracellular domains (83), IL-3 (C. Miyamoto, personal communication), interferon β (X.-X. Zhou, J. Langer, and S. Pestka, unpublished data), IL-10, vIL-10, and cmvIL-10 (L. Izotova, S. Kotenko, S. Saccani, and S. Pestka, unpublished observations).…”

Section: Protein Kinase Reactionsmentioning

confidence: 99%

Site-Specific ³² P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Clark¹,

Izotova

Philipova³

et al. 2002

BioTechniques

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Radiolabeled proteins are used in a variety of laboratory applications as well as in radioimmunotherapy. This review focuses on methods that utilize genetic engineering to introduce exogenous phosphorylation sites into proteins. Protein kinase substrate sites can be introduced into target proteins to serve as tags for several purposes. Because many protein kinases, each preferring a unique consensus sequence, are well characterized, the essential structure and function of the target protein can be effectively preserved through judicious selection and design of the phosphate incorporation site. After phosphorylation, these proteins are often indistinguishable from the parent molecules in assays of functional or biological activity. This convenient approach permits incorporation of 32P, 33P, 35S, or nonradioactive 31P, and is rapid, efficient, and safe. Most importantly, 32P labeling of monoclonal antibodies or other therapeutic protein candidates has several significant advantages over radioiodination or chemical conjugation of heavy metal isotopes.

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Section: Protein Kinase Reactionsmentioning

confidence: 99%

Site-Specific ³² P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Clark¹,

Izotova

Philipova³

et al. 2002

BioTechniques

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show abstract

“…Immunotoxins (Pastan et al, 1992;Fryxell et al, 1995;Kreitman, 1999;Rui and Chen, 2002;Urieto et al, 2004) are recombinant molecules and consist of a targeting moiety, often in the form of a monoclonal antibody or cytokine, genetically linked to a toxic moiety, usually a truncated version of diphtheria toxin (DT). A truncated DT, DT390, has been used to construct recombinant immunotoxin to avoid the nonspecific binding of DT and the inhibitory effect of preexisting anti-DT antibodies.…”

Section: Introductionmentioning

confidence: 99%

Construction and Preliminary Investigation of a Plasmid Containing a Novel ImmunotoxinDT390-IL-18Gene for the Prevention of Murine Experimental Autoimmune Encephalomyelitis

Jia

Tai

et al. 2008

DNA and Cell Biology

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Experimental autoimmune encephalomyelitis (EAE) is a neuropathological animal model for multiple sclerosis. Antigen-presenting cells (APCs) expressing interleukin-18 receptor (IL-18R) were shown to be crucial in the beginning and progress of EAE. In this study we tested the effect of a novel recombinant immunotoxin targeting IL-18R-bearing APC for EAE prevention. The novel eukaryotic plasmid DT390-IL-18-SRalpha, encoding recombinant immunotoxin DT390-IL-18, was constructed. The immunotoxin consisted of IL-18 as the targeting moiety, and a truncated diphtheria toxin (DT) as the toxic moiety. Transfection assay and proliferation inhibition assay proved the immunotoxin could be expressed in vitro and was toxic to the activated mouse T cells. To evaluate the preventive effect of DT390-IL-18-SRalpha on EAE in vivo, cationic liposome-embedded DT390-IL-18-SRalpha was injected into the hind limbs of EAE mice. DT390-IL-18-SRalpha-treated mice showed a delayed manifestation of EAE and decreased symptoms compared to the mice treated with plasmid DT390-SRalpha or phosphate-buffered saline alone. A significant reduction in infiltrating inflammatory cells was detected in the brain tissues from immunotoxin-treated mice as compared with the controls by hematoxylin-eosin staining. This study suggested that the recombinant immunotoxin DT390-IL-18 could be expressed in vitro and in vivo, and prevented murine EAE effectively.

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Introduction of Protein Kinase Recognition Sites into Proteins: A Review of Their Preparation, Advantages, and Applications

Pestka

Lin

et al. 1999

Protein Expression and Purification

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Genetic Construction of a Phosphorylation Site in Ricin A Chain: Specific Radiolabeling of Recombinant Proteins for Localization and Degradation Studies

Cited by 6 publications

References 0 publications

Site-Specific ³² P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Site-Specific ³² P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Construction and Preliminary Investigation of a Plasmid Containing a Novel ImmunotoxinDT390-IL-18Gene for the Prevention of Murine Experimental Autoimmune Encephalomyelitis

Introduction of Protein Kinase Recognition Sites into Proteins: A Review of Their Preparation, Advantages, and Applications

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Genetic Construction of a Phosphorylation Site in Ricin A Chain: Specific Radiolabeling of Recombinant Proteins for Localization and Degradation Studies

Cited by 6 publications

References 0 publications

Site-Specific 32 P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Site-Specific 32 P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Construction and Preliminary Investigation of a Plasmid Containing a Novel ImmunotoxinDT390-IL-18Gene for the Prevention of Murine Experimental Autoimmune Encephalomyelitis

Introduction of Protein Kinase Recognition Sites into Proteins: A Review of Their Preparation, Advantages, and Applications

Contact Info

Product

Resources

About

Site-Specific ³² P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Site-Specific ³² P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application