Yun Bai1, Wei-Chiang Shen. 1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.
Abstract
PURPOSE: To improve the oral efficacy of the recombinant fusion protein containing granulocyte colony-stimulating factor (G-CSF) and transferrin (Tf) by inserting a linker between the two protein domains. MATERIALS AND METHODS: Oligonucleotides encoding flexible and helix-forming peptides were inserted to the recombinant plasmids. The fusion protein without linker insertion was used for comparison. The G-CSF cell-proliferation and Tf receptor-binding activities of the fusion proteins were tested in NFS-60 cells and Caco-2 cells, respectively, and in vivo myelopoietic assay with both subcutaneous and oral administration was performed in BDF1 mice. RESULTS: All fusion proteins produced from transfected HEK293 cells were positive in Western-blotting assay with anti-G-CSF and anti-Tf antibodies. Among them, the fusion protein with a long helical (H4-2) linker showed the highest activity in NFS-60 cell proliferation assay, with an EC50 about ten-fold lower than that of the non-linker fusion protein. The fusion protein with H4-2 linker also showed a significantly higher myelopoietic effect when administered either subcutaneously or orally in BDF1 mice. CONCLUSION: The insertion of a linker peptide, such as the helix linker H4-2, between G-CSF and Tf domains in the recombinant fusion protein can improve significantly both in vitro and in vivo myelopoietic activity over the non-linker fusion protein.
PURPOSE: To improve the oral efficacy of the recombinant fusion protein containing granulocyte colony-stimulating factor (G-CSF) and transferrin (Tf) by inserting a linker between the two protein domains. MATERIALS AND METHODS:Oligonucleotides encoding flexible and helix-forming peptides were inserted to the recombinant plasmids. The fusion protein without linker insertion was used for comparison. The G-CSF cell-proliferation and Tf receptor-binding activities of the fusion proteins were tested in NFS-60 cells and Caco-2 cells, respectively, and in vivo myelopoietic assay with both subcutaneous and oral administration was performed in BDF1 mice. RESULTS: All fusion proteins produced from transfected HEK293 cells were positive in Western-blotting assay with anti-G-CSF and anti-Tf antibodies. Among them, the fusion protein with a long helical (H4-2) linker showed the highest activity in NFS-60 cell proliferation assay, with an EC50 about ten-fold lower than that of the non-linker fusion protein. The fusion protein with H4-2 linker also showed a significantly higher myelopoietic effect when administered either subcutaneously or orally in BDF1 mice. CONCLUSION: The insertion of a linker peptide, such as the helix linker H4-2, between G-CSF and Tf domains in the recombinant fusion protein can improve significantly both in vitro and in vivo myelopoietic activity over the non-linker fusion protein.
Authors: Teresa Burgess; Angela Coxon; Susanne Meyer; Jan Sun; Karen Rex; Trace Tsuruda; Qing Chen; Shu-Yin Ho; Luke Li; Stephen Kaufman; Kevin McDorman; Russell C Cattley; Jilin Sun; Gary Elliott; Ke Zhang; Xiao Feng; Xiao-Chi Jia; Larry Green; Robert Radinsky; Richard Kendall Journal: Cancer Res Date: 2006-02-01 Impact factor: 12.701
Authors: Michael Koenigsmann; Kathleen Jentsch-Ullrich; Martin Mohren; Elke Becker; Marcell Heim; Astrid Franke Journal: Transfusion Date: 2004-05 Impact factor: 3.157
Authors: Vladimir L Kolossov; Bryan Q Spring; Robert M Clegg; Jennifer J Henry; Anna Sokolowski; Paul J A Kenis; H Rex Gaskins Journal: Exp Biol Med (Maywood) Date: 2011-05-23