Human Tumor Necrosis Factor-alpha, Mutant (Recombinant)

Recombinant Human Tumor Necrosis Factor-alpha Mutant

Source: E.Coli

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-alfa 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-alfa 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-a Variant produced in E.Coli is a single, non-glycosylated, polypeptide chain containing 151 amino acids and having a molecular mass of 16598 Dalton. The Human TNF-alpha Variant is purified by standard chromatographic techniques.

Physical Appearance: Sterile Filtered White lyophilized (freeze-dried) powder.

Formulation: The protein was lyophilized after extensive dialysis against 0.5x PBS pH -7.

Solubility: It is recommended to reconstitute the lyophilized Recombinant Human TNF-alpha Variant in sterile 18MO-cm H2O not less than 100 g/ml, which can then be further diluted to other aqueous solutions.

Stability: Lyophilized Recombinant Human TNF-alpha Variant although stable at room temperature for 3 weeks, should be stored desiccated below -180C. Upon reconstitution Human TNF-alpha Variant should be stored at 40C between 2-7 days and for future use below -180C. For long term storage it is recommended to add a carrier protein (0.1% HSA or BSA). Please prevent freeze-thaw cycles.

Purity: Greater than 95.0% as determined by: (a) Analysis by RP-HPLC. (b) Anion-exchange FPLC. (c) 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 MRKRKPVAHV VANPQAEGQL QWLNRRANAL LANGVELRDN QLVVPSEGLY LIYSQVLFKG QGCPSTHVLL THTISRIAVS YQTKVNLLSA IKSPCQRETP EGAEAKPWYE PIYLGGVFQL EKGDRLSAEI NRPDYLDFAE SGQVYFGIIAF.

Dimers and aggregates: Less than 1% as determined by silver-stained SDS-PAGE gel analysis.

Biological Activity: ProSpec's Recombinant Human TNF-alpha is Variant fully biologically active when compared to the wild type. The ED50 as determined by the cytolysis of murine L929 cells in the presence of Actinomycin D is < 0.05ng/ml, corresponding to a Specific Activity of 1 x 108 IU/mg.

Endotoxin: Less than 0.1 ng/ g (IEU/ g) of Human TNF-alpha.

Protein content: Protein quantitation was carried out by two independent methods: 1. UV spectroscopy at 280 nm. 2. Analysis by RP-HPLC, using a calibrated solution of TNF-alfa as a Reference Standard.

Usage: BioWORLD's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.