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Clonagen

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Recombinant Human Tumor Necrosis Factor alpha Mutant (TNFa Mutant)
(Cat. No.: C036)


Background:
        TNF is secreted by macrophages, monocytes, neutrophils, T-cells, NK-cells following their stimulation by bacterial LPS. Cells expressing CD4 secrete TNF-alpha while CD8 cells secrete little or no TNF-alpha. The synthesis of TNF-alpha is induced by many different stimuli including interferons, IL2, GM-CSF.

Description:
       
The clinical use of the potent anti-tumor activity of TNF-alpha has been limited by the proinflammatory side effects including fever, dose-limiting hypotension, hepatotoxicity, intravascular thrombosis, and hemorrhage. Designing clinically applicable TNF-a mutants with low systemic toxicity has been an intense pharmacological interest. Human TNF-a, which binds to the murine TNF-R55 but not to the mouse TNF-R75, exhibits retained anti-tumor activity and reduced systemic toxicity in mice compared with murine TNF-a, which binds to both murine TNF receptors. Based on these results, many TNF-a mutants that selectively bind to TNF-R55 have been designed. These mutants displayed cytotoxic activities on tumor cell lines in vitro, and exhibited lower systemic toxicity in vivo. Recombinant Human TNF-alpha Variant/Mutant compared with the wild-type, has an amino acid sequence deletion from a.a. 1-7, and the following a.a. substitutes Arg8, Lys9, Arg10 and Phe157 which is proven to have more activity and with less inflammatory side effect in vivo. Recombinant Human TNF-alpha Mutant produced in E. coli is a single, non-glycosylated, polypeptide chain containing 151 amino acids and having a molecular mass of 16,886 Dalton.

Quality Control:
        Biological activity:
The ED50 as determined by the cytolysis of murine L929 cells in the presence of Actinomycin D is less then 0.01 ng/ml, corresponding to a Specific Activity of 1.0 x 108 IU/mg.
        Purity: Greater than 95% as determined by
                    (a) Analysis by SEC-HPLC.
                    (b) Analysis by reducing and non-reducing SDS-PAGE Silver Stained gel.
        Amino-Acid Sequence: The sequence of the first five N-terminal amino acids was determined and was found to be Met-Arg-Lys-Arg-Lys.
        Endotoxin: Less than 0.01ng/µg (0.01IEU/µg) determined by LAL test.

Formulation: Lyophilized from a 0.2 µm filtered solution in PBS.

Storage: Lyophilized samples are stable for up to twelve months from date of receipt at -20oC to -70oC.
        Please avoid freeze-thaw cycles.

Reconstitution: It is recommended to reconstitute the lyophilized TNF-alpha Mutant in sterile 18MΩ-cm H2O not less than 100µg/ml, which can then be further diluted to other aqueous solutions.

 

 


 
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Last modified: 05/29/09