In vitro activity of Fc-FGF21(RG) relative to native FGF21 and Fc-FGF21 wild type.

<p>(A) Structural diagram of Fc-FGF21(RG), an E coli-expressed homodimeric Fc-FGF21 fusion protein containing 2 engineered point mutations at positions 98 and 171. 15G indicates the 15 aa polyglycine and polyserine linker. (B) The binding activity of FGF21, Fc-FGF21 and Fc-FGF21(RG) to human βKlotho determined in a solution equilibrium binding assay on a BIAcore instrument. (C) Elk1-luciferase reporter activity in 293T cells treated with FGF1, FGF21, Fc-FGF21 and Fc-FGF21(RG). The cells were stably expressing reporter constructs but no βKlotho. (D) Stimulation of Elk1-luciferase reporter activity by FGF21, Fc-FGF21 and Fc-FGF21(RG) in 293T cells stably expressing human βKlotho along with luciferase reporter constructs. (E) Stimulation of Elk1-luciferase reporter activity by FGF21, Fc-FGF21 and Fc-FGF21(RG) in mouse NIH/3T3 fibroblast cells transiently co-transfected with human βKlotho and human FGFR1c. (F) Stimulation of Erk phosphorylation by FGF21, Fc-FGF21 and Fc-FGF21(RG) in human primary adipocytes. All data represent mean ± SEM, n = 4/concentration group. ND: not detectable. FGF21, closed circle; Fc-FGF21: opened circle; Fc-FGF21(RG): closed triangular and dash line; FGF1: closed square.