TNF Alpha^HuXp Authentic Trimer Human Cell Expressed

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Introduction

Cytokines are a group of proteins and polypeptides that organisms use as signaling molecules. Most cytokines are glycoproteins less than 30 kDa in size and bind to specific, high-affinity cell surface receptors. Due to their central role in the immune system, cytokines are involved in a variety of immunological, inflammatory and infectious diseases and widely used in research, diagnostics and therapeutics. Cytokines generally alter the gene expression pattern of the target cell which leads to changes in the rate of cell proliferation and/or in the state of cell differentiation. Currently, these proteins are predominantly produced in non-human cells (e.g. E coli, SF9, CHO ) and therefore lack authenticity due to the absence of physiologically relevant glycosylation. In addition, a number of important cytokines are not commercially available due to inadequate proteolytic processing, protein folding or other post-translational modifications that occur in the non-human cell expression systems. HumanZyme has developed an efficient human-cell based technology, HumaXpress™ for scalable production of human cytokines.

TNF alpha

TNF alpha is a member of the prototypic ligand of the TNF superfamily. This cytokine plays a central role in inflammation, apoptosis, and immune system development. The native 26 kD transmembrane protein is assembled intracellularly to form a noncovalently linked homotrimer¹. Cleavage of the membrane bound TNF alpha by TACE/ADAM17 releases a 55 kD soluble trimeric form that regulates lymphoid tissue development and promotes inflammation responses².

Other commercially available TNF alpha proteins are produced from E coli. HumanZyme has produced TNF alpha^HuXp from engineered human 293 cells. Both E. coli and human cell expressed proteins exhibit a molecular mass of 18 kD in SDS-PAGE. However, gel filtration studies indicate that only TNF alpha^HuXp is trimeric, which has a molecular mass of 51kD, while the E coli form is not a trimer. These proteins also show distinct mobility on native gel electrophoresis.

The bioactivity of TNF alphaHuXp was determined by the dose-dependent cytotoxity of the TNF alpha sensitive cell line L-929 in the presence of Actinomycin D. The biological effect was also compared with RA synoviocytes. 500,000 cells/wells were used in 6 well plates. Cells were starved for 12h with DMEM media without FCS. Cells were then stimulated for 24h in 2 ml of DMEM without FCS. The IL6 production was analyzed by ELISA on the supernatants. The data in the figure below indicate that TNF alphaHuXp is more effective than the E coli-expressed cytokine.

TNF alphaHuXp: Higher Efficacy-Production of IL-6 in Rheumatoid Synoviocytes

(View more information for product number HZ-1014.)

TNF alpha^HuXp is available in trial size and in bulk.

HumanZyme has developed and continues to develop a growing range of tag-free cytokines, including difficult-to-express protein members of the TGFβ superfamily. HumanZyme Authentic Cytokines can be used as highly preferred reagents in a wide range of applications for cancer, inflammation, stem cell research, and antibody development. References

1.Tang, P et al., 1996 Biochemistry 35:8216
2. Black, R.A et al., 1997, Nature 385:729