SDS-PAGE analysis of the localization and purification of an expressed GEM protein. Bands indicated by arrows correspond to thioredoxin-hGrnA fusion protein (fusion, ∼25.8 kDa), thioredoxin tag protein fragment (Trx, ∼17.9 kDa), and granulin A (Grn, ∼7.9 kDa). (A) IPTG-induced Origami(DE3) cells transformed with the granulin A construct after sonication for 3, 4, and 5 min. Soluble (3S, 4S, and 5S) and insoluble fractions (3P, 4P, and 5P) were separated by centrifugation, transferred into an equal volume of a solution that was 55 mM in Tris-HCl, 70 mM DTT, 2.2% SDS, 10% glycerol, pH 6.8, and analyzed by SDS-PAGE. (B) Purification of the thioredoxin-hGrnA fusion protein on a Ni-NTA agarose column. Protein fractions were eluted progressively with a solution of 8 M urea in 0.1 M sodium phosphate buffer, 0.01 M Tris-HCl pH 6.3 (lane I), pH 5.9 (lane II), and pH 4.5 (lane III). (C) Release of the GEM peptide by enterokinase cleavage of the thioredoxin-hGrnA fusion protein. (D) Partial purification of the mixture of Grn disulfide isomers. The reaction mixture after enterokinase cleavage containing granulin A was applied onto a Waters Sep-Pak C18 cartridge and washed as specified in Materials and Methods. Granulin A was eluted with three column volumes of 35% acetonitrile.