The Experts below are selected from a list of 12 Experts worldwide ranked by ideXlab platform
Jp Mckearn - One of the best experts on this subject based on the ideXlab platform.
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Phage display mutagenesis of the chimeric dual Cytokine Receptor Agonist myelopoietin
Leukemia, 2001Co-Authors: R Ibdah, C Van Valkenburgh, E Rowold, A Abegg, A Donnelly, J Klover, S Merlin, Jp MckearnAbstract:Myelopoietins comprise a class of chimeric Cytokine Receptor Agonists consisting of an hIL-3 (human interleukin-3) Receptor Agonist and an hG-CSF (human granulocyte colony-stimulating factor) Receptor Agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting Cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component Agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding Receptor. Five amino acid positions in a short region of the hG-CSF Receptor Agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very ‘deep’ mutagenesis at selected residues: >10^5 substitution variants were affinity-screened using the hG-CSF Receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.
R Ibdah - One of the best experts on this subject based on the ideXlab platform.
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Phage display mutagenesis of the chimeric dual Cytokine Receptor Agonist myelopoietin
Leukemia, 2001Co-Authors: R Ibdah, C Van Valkenburgh, E Rowold, A Abegg, A Donnelly, J Klover, S Merlin, Jp MckearnAbstract:Myelopoietins comprise a class of chimeric Cytokine Receptor Agonists consisting of an hIL-3 (human interleukin-3) Receptor Agonist and an hG-CSF (human granulocyte colony-stimulating factor) Receptor Agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting Cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component Agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding Receptor. Five amino acid positions in a short region of the hG-CSF Receptor Agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very ‘deep’ mutagenesis at selected residues: >10^5 substitution variants were affinity-screened using the hG-CSF Receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.
Joseph K. Welply - One of the best experts on this subject based on the ideXlab platform.
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Mammalian cell production and purification of progenipoietin, a dual‐Agonist chimaeric haematopoietic growth factor
Biotechnology and Applied Biochemistry, 2003Co-Authors: David C. Wood, Karl J. Mathis, William D. Joy, Minnerly John C, Lyle E. Pegg, Joseph K. WelplyAbstract:One member of the progenipoietin (ProGP) family of engineered proteins, ProGP-2, is a chimaeric dual Cytokine Receptor Agonist, expressed in mammalian cells, that stimulates both human fetal liver tyrosine kinase-3 (Flt3) and the granulocyte-colony-stimulating-factor (G-CSF) Receptor. The production of ProGP-2 on a small and large scale using either anti-(Flt3 ligand) antibody-affinity chromatography, or a combination of (NH 4 ) 2 SO 4 fractionation, anion-exchange chromatography, hydrophobic-interaction chromatography and preparative reverse-phase chromatography is described. ProGP-2 was produced in hollow-fibre reactors containing stably transfected NS0 cells. The productivity of ProGP-2 was initially high, but was found to decrease 3-4-fold over time. When the yield of ProGP-2 decreased, the combination of three conventional chromatography steps was required to meet protein purity similar to that achieved by the anti-(Flt3 ligand) chromatography method. In addition, a protease activity was observed in conditioned media from the hollow-fibre reactors that resulted in increased degradation of ProGP-2 that was removed by hydrophobic-interaction chromatography at higher pH. Together the results demonstrated a method for production and purification of ProGP-2 for additional studies on its haematopoietic activity.
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Mammalian cell production and purification of progenipoietin, a dual-Agonist chimaeric haematopoietic growth factor.
Biotechnology and applied biochemistry, 2003Co-Authors: David C. Wood, Karl J. Mathis, William D. Joy, Lyle E. Pegg, John C Minnerly, Joseph K. WelplyAbstract:One member of the progenipoietin (ProGP) family of engineered proteins, ProGP-2, is a chimaeric dual Cytokine Receptor Agonist, expressed in mammalian cells, that stimulates both human fetal liver tyrosine kinase-3 (Flt3) and the granulocyte-colony-stimulating-factor (G-CSF) Receptor. The production of ProGP-2 on a small and large scale using either anti-(Flt3 ligand) antibody-affinity chromatography, or a combination of (NH4)2SO4 fractionation, anion-exchange chromatography, hydrophobic-interaction chromatography and preparative reverse-phase chromatography is described. ProGP-2 was produced in hollow-fibre reactors containing stably transfected NS0 cells. The productivity of ProGP-2 was initially high, but was found to decrease 3-4-fold over time. When the yield of ProGP-2 decreased, the combination of three conventional chromatography steps was required to meet protein purity similar to that achieved by the anti-(Flt3 ligand) chromatography method. In addition, a protease activity was observed in conditioned media from the hollow-fibre reactors that resulted in increased degradation of ProGP-2 that was removed by hydrophobic-interaction chromatography at higher pH. Together the results demonstrated a method for production and purification of ProGP-2 for additional studies on its haematopoietic activity.
C Van Valkenburgh - One of the best experts on this subject based on the ideXlab platform.
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Phage display mutagenesis of the chimeric dual Cytokine Receptor Agonist myelopoietin
Leukemia, 2001Co-Authors: R Ibdah, C Van Valkenburgh, E Rowold, A Abegg, A Donnelly, J Klover, S Merlin, Jp MckearnAbstract:Myelopoietins comprise a class of chimeric Cytokine Receptor Agonists consisting of an hIL-3 (human interleukin-3) Receptor Agonist and an hG-CSF (human granulocyte colony-stimulating factor) Receptor Agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting Cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component Agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding Receptor. Five amino acid positions in a short region of the hG-CSF Receptor Agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very ‘deep’ mutagenesis at selected residues: >10^5 substitution variants were affinity-screened using the hG-CSF Receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.
E Rowold - One of the best experts on this subject based on the ideXlab platform.
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Phage display mutagenesis of the chimeric dual Cytokine Receptor Agonist myelopoietin
Leukemia, 2001Co-Authors: R Ibdah, C Van Valkenburgh, E Rowold, A Abegg, A Donnelly, J Klover, S Merlin, Jp MckearnAbstract:Myelopoietins comprise a class of chimeric Cytokine Receptor Agonists consisting of an hIL-3 (human interleukin-3) Receptor Agonist and an hG-CSF (human granulocyte colony-stimulating factor) Receptor Agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting Cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component Agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding Receptor. Five amino acid positions in a short region of the hG-CSF Receptor Agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very ‘deep’ mutagenesis at selected residues: >10^5 substitution variants were affinity-screened using the hG-CSF Receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.
