ISOLATION OF ERYTHROPOIETIN RECEPTOR AGONIST PEPTIDES USING EVOLVED PHAGE LIBRARIES

Cyclic peptides capable of activating the erythropoietin receptor (EPO R) were isolated from phage display libraries by screening with a nove l EPOR-IgG fusion protein reagent. A parental clone ERB1 (EPO Receptor Binder 1) was first isolated from a phage display library displaying 38 random amino acids as an N-terminal fusion with the M13 minor capsi d protein, pill. An evolved library was then produced from the parenta l sequence using an oligonucleotide saturation mutagenesis strategy wh ich yielded EPOR binding sequences with 20 times the relative affinity of ERB1, Two synthetic peptides were constructed from these sequences both of which bind the EPO receptor in specific ELISA, and act as ful l agonists in EPO dependent cell proliferation assays. These peptides are 18 amino acids in length, disulfide-bonded, and have a minimum con sensus sequence of CXXGWVGXCXXW, where X represents positions tolerant of several amino acids.