Ortho Pharmaceutical Corporation

Ortho Diagnostic Systems Inc.

Frank J McMahon

Steven A Middleton

Linda K Jolliffe

Leopoldo G Mendoza

Chander Bahl

Dana Johnson

Linda S Mulkahy

The extracellular domain of the human erythropoietin receptor (EPO binding protein, EBP) has been expressed and overproduced in,. 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 &lsqb;,I&rsqb;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 EBP-Ig, is a preformed dimerization template and is also useful in drug design and discovery methods.

Methods for identifying erythropoietin receptor binding protein

The present invention provides labeled nucleotide probes and a procedure for synthesis of the labeled nucleotide probe by a coupling or joinder of two components of the probe, namely a target recognition sequence moiety and a signal generating moiety, through the use of a bridging complement that holds the two moieties in such a position as to allow formation of a sugar-phosphate chemical linkage between the 3' end of one moiety and the 5' end of the other moiety.

Method of preparing nucleotide probes using a bridging complement

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Whitehouse Station, NJ, United States of America

Frank J McMahon