Journal of Zhejiang University SCIENCE
(ISSN 1009-3095, Monthly)
2003   Vol. 4   No. 3   p.340-345

Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-Receptor and identification of its activity

SUN Yi(孙益)(College of Life Sciences, Zhejiang University, Hangzhou 310027, China)
Marz Pia(Department of Physiology, Basel University, Vesalgasse 1, 4051 Basel, Switzerland)
Otten Uwe(Department of Physiology, Basel University, Vesalgasse 1, 4051 Basel, Switzerland)
GE Ji-guang(葛霁光)(College of Life Sciences, Zhejiang University, Hangzhou 310027, China)
Rose-John Stefan(Department of Biochemistry, Christian-Albrechts University of Kiel, D-24098 Kiel, Germany)

Abstract:Ciliary neurotrophic factor (CNTF) has pleiotropic actions on many neuronal populations as well as on glia. Signal transduction by CNTF requires that it bind first to CNTF-R, permitting the recruitment of gp130 and LIF-R, forming a tripartite receptor complex. Cells that only express gp130 and LIF-R, but not CNTF-R are refractory to stimulation by CNTF. On many target cells CNTF only acts in the presence of its specific agonistic soluble receptors. We engineered a soluble fusion protein by linking the COOH-terminus of sCNTF-R to the NH2 -terminus of CNTF. Recombinant CNTF/sCNTF-R fusion protein (Hyper-CNTF) was successfully expressed in COS-7 cells. The apparent molecular mass of the Hyper-CNTF protein was estimated from western blots to be 75 kDa. Proliferation assays of transfected BAF/3 cells in response to CNTF and Hyper-CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gp130, CNTF-R and LIF-R receptor subunit whereas Hyper-CNTF required heterodimerization of the gp130 and LIF-R receptor subunit. We concluded that the fusion protein Hyper-CNTF had superagonistic activity on target cells expressing gp130 and LIF-R, but lacking membrane-bound CNTF-R.
Keywords:Ciliary neurotrophic factor(CNTF), Soluble CNTF-Receptor, Fusion protein, Biological activity

CLC Number:Q78 Document ID:A

:Foundation Item:Project supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholar, Zhejiang Province, China and SFB415, TP B5, Germany.
:Author Resume:SUN Yi,E-mail: sun-yi@mail.hz.zj.cn

:References:

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:Manuscript Received:2002年6月14日

:Manuscript Revised:2002年9月29日

Published:2003年6月1日