Enhanced signal transduction by a directly fused protein of interleukin-6 and its receptor.

In order to develop a new type of agonist for the interleukin 6 (IL-6) signal, the gene encoding a directly fused protein (DFP) was constructed by joining the N-terminal portion of IL-6 and the C-terminal (soluble) portion of IL-6R (sIL-6R) without using a flexible polypeptide linker. The biological activity if DFP from a recombinant Pichia pastoris was examined by growth stimulation of IL-6-dependent BAF130 cells expressing human gp130, a membrane receptor. The recombinant DFP exhibited a much stronger growth stimulation (10 times) than the independent IL-6 and sIL-6R (IL-6/sIL-6R), mainly because association of the IL-6 and IL-6R could be maintained even at lower concentrations of DFP. Surface plasmon resonance (SPR) analysis showed that DFP bound to the extracellular portion of gp130 in the biphasic mode, and the dissociation constants of DFP for two phases were the same as those of IL-6/sIL-6R. In cells treated with DFP, stimulation of Stat3 phosphorylation was maintained for a longer period (150 min) than in cells treated with IL6/Il-6R, suggesting that the signal mediated by the DFP was more durable than that mediated by IL-6/sIL-6R, although the signal transduction mechanisms are almost the same for both DFP and IL-6/IL-6R. Therefore, the stronger activity of DFP was attributed to the maintained association of its subunits and/or prolonged phosphorylation of Stat3.

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