Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Bai
Yun
Y
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.
Shen
Wei-Chiang
WC
D000284:Administration, Oral; D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D016179:Granulocyte Colony-Stimulating Factor / Q000008:administration & dosage / Q000737:chemistry* / Q000493:pharmacokinetics*; D007279:Injections, Subcutaneous; D008297:Male; D051379:Mice; D038042:Myelopoiesis / Q000187:drug effects; D010455:Peptides / Q000737:chemistry; D010957:Plasmids; D011990:Receptors, Transferrin / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D014168:Transferrin / Q000008:administration & dosage / Q000737:chemistry* / Q000493:pharmacokinetics*
D010455:Peptides; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D016179:Granulocyte Colony-Stimulating Factor