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

Document Type

Article

Publication Date

2006

Abstract

Purpose. To improve the oral efficacy of the recombinant fusion protein containing granulocyte colonystimulating 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.

Publication Title

Pharmaceutical research

Volume

23

Issue

9

First Page

2116

Last Page

2121

Comments

This article was published in Pharmaceutical research, Volume 23, Issue 9, Pages 2116-2121.

The published version is available at http://dx.doi.org/10.1007/s11095-006-9059-5.

Copyright © 2006 Springer.

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