Quick method of multimeric protein production for biologically active substances such as human GM-CSF (hGM-CSF).
The C-terminal fragment of C4b-binding protein (C4BP)-based multimerizing system was applied to hGM-CSF to induce dendritic cells (DCs) from peripheral blood monocytes (PBMCs), to see whether the C4BP could stimulate immature DCs, since DCs, equipped with pattern recognition receptors such as toll-like receptors (TLRs), are hypersensitive to various immunologically active molecules like LPS. hGM-CSF gene was merged to the 3'-terminal region of the C4BPalpha-chain gene, and the transfected human 293FT cells produced sufficient amount of octameric hGM-CSF, which resulted in iDCs with the same phenotype and the same response to a TRL4 ligand, LPS and a TLR3 ligand, poly I:C, as those induced with authentic monomeric hGM-CSF. These results suggest that the C4BP-based multimerizing system could facilitate the design of self-associating multimeric recombinant proteins without stimulating iDCs, which might be seen with the other multimerizing systems such as that using Fc fragment of IgM.
Shinya
Eiji
E
Department of Microbiology and Immunology, Nippon Medical School, Graduate School 2D03, Bunkyo City, Tokyo 113-8602, Japan. eiji@nms.ac.jp
Owaki
Atsuko
A
Norose
Yoshihiko
Y
Sato
Shigeru
S
Takahashi
Hidemi
H
D002460:Cell Line; D050716:Complement C4b-Binding Protein / Q000235:genetics / Q000378:metabolism; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D014176:Protein Biosynthesis; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
D050716:Complement C4b-Binding Protein; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor