C - Chemistry – Metallurgy – 07 – K
Patent
C - Chemistry, Metallurgy
07
K
C07K 14/71 (2006.01) G01N 33/566 (2006.01) C07K 14/505 (2006.01)
Patent
CA 2573196
The extracellular domain of the human erythropoietin receptor (EPO binding protein, EBP) bas been expressed and overproduced in E. coli. Control of oxygen levels and pH during high density fermentation allows the production of only the protein variant with the native amino terminus. Methods disclosed permit the efficient recovery of purified EBP which quantitatively binds EPO. The active purified protein competes with membrane associated EPO receptor for binding [125]EPO and neutralizes EPO dependent stimulation in a cell based proliferation assay. Further, the radioligand equilibrium binding constant for this interaction has been determined by immobilizing EBP on agarose gel via a free cysteine. The EBP of the present invention has many uses including the structural determination of the protein by NMR or crystallography, in drug design and discovery, and as a therapeutic. A fusion protein of EBP and an immunoglobulin heavy chain was also produced. This protein, termed IBP-Ig, is a preformed dimerization template and is also useful in drug design and discovery methods.
Johnson Dana
Jolliffe Linda K.
Mcmahon Frank
Middleton Steven A.
Mulkahy Linda S.
Ortho-Mcneil Pharmaceutical Inc.
Smart & Biggar
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