Fusion protein of interleukin-6 and interleukin-6 receptor without a polypeptide linker.
FP6, a novel recombinant fusion protein of interleukin-6 (IL-6) and IL-6 receptor (IL-6R), was prepared in the methylotrophic yeast Pichia pastoris. This protein was a potent activator of a cell surface transducing glycoprotein, gp130 and is a potential therapeutical reagent in the hemopoietic field. A linker is generally thought to be required for two fused molecules to retain their proper structures although it should preferably be removed to reduce possible antigenicity. It was found that the C-terminal residue of IL-6R could be directly linked to the N-terminal residue of IL-6 without decreasing the ability of IL-6 to bind gp130 and send the IL-6 signal. It was also found that the peptide bond between Lys-37 and Asp-38 of IL-6 was prone to proteolytic cleavage and that the immunoglobulin (Ig)-like region of IL-6R underwent extensive and heterogeneous glycosylation when expressed in P. pastoris. Based on these findings, we designed FP6 without the Ig-like region, in which the C-terminal residue of Ala-333 of IL-6R was directly linked to Asp-38 of IL-6 by a peptide bond. Purified FP6 had both an in vitro effect on hemopoietic progenitors to generate various colonies and an in vivo effect on megakaryocyte progenitors to increase platelet counts. Four purified FP6s were obtained, which had the same molecular mass and different isoelectric points without any detectable modification in the course of purification. The difference in isoelectric points was shown to be due to microheterogeneity of the carbohydrate chains. Each FP6 had the same specific activity in the cell growth assay with or without endoglycosidase digestion. Homogeneous FP6 with respect to isoelectric point as well as molecular mass merits more detailed characterization and evaluation for possible clinical application.
Yasukawa
Kiyoshi
K
Tokyo Research Loboratories, Tosoh Corporation, 2743-1 Hayakawa, Ayase-shi, Kanagawa 252-1123, Japan. yasukawa@tosoh.co.jp
Tsuchiya
Shigeo
S
Ekida
Teiji
T
Iida
Hiroshi
H
Ide
Teruhiko
T
Miki
Daisuke
D
Yagame
Harutaka
H
Murayama
Keiichi
K
Ishiguro
Takahiko
T