Figure 1.

Production of a secreted full-length extracellular domain of Klotho in mammalian cells. A, amino acid sequence of a secreted full-length ectodomain of human Klotho. The KL1 domain is shown in violet, and the KL2 domain is green. Putative N-linked sites are blue. V5 tag and His₆ tag are black. Four residues at highly conserved positions are red. B, SDS-PAGE analysis of purified sKlotho protein expressed in mammalian cells. sKlotho protein in A was expressed in either stable CHO or transient HEK293 and purified as described under “Experimental procedures.” A spliced region in the gel is indicated with a heavy line. C, IEF gel analysis of purified sKlotho protein. D, SEC analysis of purified sKlotho protein. Bio-Rad gel filtration standards indicated by blue arrows are thyroglobulin (bovine, 670 kDa), γ-globulin (bovine, 158 kDa), ovalbumin (chicken, 44 kDa), myoglobin (horse, 17 kDa), and vitamin B₁₂ (1.35 kDa).