| 主权项 |
1. A method of cell culture, comprising the steps of
(i) obtaining a stem or progenitor cell sample, (ii) culturing the stem or progenitor cell sample in media and under closed conditions appropriate to cause proliferation or differentiation of the stem or progenitor cells, and (iii) optionally purifying the stem or progenitor cells ex vivo, wherein the media comprises a vEPO protein variant selected from the group consisting of:
(a) proteins termed hs3, h1-4, h1-5, hs4, h1-I, h2-1, mS, mG3, mG5, m301 and mK3 having the deduced amino acid sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, respectively;(b) proteins encoded by polynucleotides having the coding sequence as of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 encoding at least the mature form of the protein;(c) proteins encoded by a polynucleotide encoding a humanized version of the proteins mS, mG3, mG5, m301 and mK3 having the deduced amino acid sequence of SEQ ID NOs 14, 16, 18, 20, and 22;(d) a protein comprising a fusion of an amino acid sequence selected from the group of amino acid sequences of SEQ ID NO 24, 26, 28, and 30, at the N-terminus of an amino acid sequence selected from the group of amino acid sequences of SEQ ID NO 32, 34, 36, and 38;(e) a protein encoded by a polynucleotide comprising a fusion of polynucleotide sequences selected from the group of polynucleotide sequences of SEQ ID NO 23, 25, 27, and 29, 5′ of a polynucleotide sequence selected from the group of polynucleotide sequences of SEQ ID NO 31, 33, 35, and 37;(f) a derivative of a protein or a peptide encoded by a polynucleotide of anyone of (a) to (e), wherein in said derivative between 1 and 10 amino acid residues are conservatively substituted compared to said protein, and said derivative has neuroprotective activity, but essentially no hematopoietic activity;(g) the protein of (a) to (f), wherein in said fragment between 1 and 10 amino acid residues are N- and/or C-terminally deleted and/or between 1 and 10 amino acids are deleted N- and or C-terminally of the junction compared to said protein, and said fragment has neuroprotective activity, but essentially no hematopoietic activity;(h) a protein termed ha, hAmA, hAmE, hA-10 and hA-10-transport, hA-transport sequence, having the deduced amino acid sequence of SEQ ID NOs 50, 51, 52, 53, 61 and 66 respectively;(i) a protein encoded by a polynucleotide having the coding sequence of SEQ ID NOs: 55, 56, 57, 58, 60 and 65 encoding at least the mature form of the protein;(j) a derivative of a protein termed ha, hAmA, hAmE and hA-10, or a protein encoded by a polynucleotide having the coding sequence of SEQ ID NOs: 55, 56, 57 and 58 encoding at least the mature form of the protein, wherein in said derivative between 1 and 10 amino acid residues are conservatively substituted compared to said protein, and said derivative has neuroprotective activity, but essentially no hematopoietic activity;(k) a fragment of a protein termed ha, hAmA, hAmE and hA-10, or a protein encoded by a polynucleotide having the coding sequence of SEQ ID NOs: 55, 56, 57 and 58 encoding at least the mature form of the protein, wherein in said fragment between 1 and 10 amino acid residues are N- and/or C-terminally deleted and/or between 1 and 10 amino acids are deleted N- and or C-terminally of the junction compared to said protein, and said fragment has neuroprotective activity, but essentially no hematopoietic activity;(l) an erythropoietin (EPO) variant encoded by a polynucleotide selected from the group consisting of:
(1) polynucleotides encoding the N-terminal part of full length EPO including helix A and which lack at least one of the following:(i) a fragment of at least 10 amino acids between helix A and helix B,(ii) a fragment of at least 10 amino acids of helix B,(iii) a fragment of at least 2 amino acids between helix B and helix C,(iv) a fragment of at least 10 amino acids of helix C,(v) a fragment of at least 10 amino acids between helix C and D, and/or(vi) a fragment of at least 10 amino acids of helix D, wherein said variant has neuroprotective activity, but essentially no hematopoietic activity,(2) polynucleotides encoding a derivative of a protein encoded by a polynucleotide of anyone of (a), wherein in said derivative between 1 and 10 amino acid residues are conservatively substituted compared to said protein, and said derivative has neuroprotective activity, but essentially no hematopoietic activity and,(3) polynucleotides, the complementary strand of which hybridizes under stringent conditions to a polynucleotide as defined in (1) and which code for a protein having neuroprotective activity, but essentially no hematopoietic activity; or the complementary strand of such a polynucleotide;(m) a derivative of a protein of anyone of (a) to (l), wherein in said derivative amino acid residues are truncated from the transport sequence (SEQ ID NO 62 for amino acid sequence and SEQ ID NO 63 for polynucleotide sequence), and said derivative has neuroprotective activity, but essentially no hematopoietic activity. |
